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Liu Y, Jiang Y, Ma T, Dong W, Yang P, Peng L, Wang B, Wu C, Li Z, Zhang H, Sun Y, Niu Y, Ding Y. Cardiomyocyte-specific activation of the sarcomere-localized Dnajb6b chaperone causes cardiomyopathy and heart failure through upregulated sarcoplasmic reticulum stress. Life Sci 2025; 374:123711. [PMID: 40360088 DOI: 10.1016/j.lfs.2025.123711] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2025] [Revised: 05/01/2025] [Accepted: 05/09/2025] [Indexed: 05/15/2025]
Abstract
AIMS Despite abundant expression of DNAJB6 gene in the heart, its roles in cardiac diseases remain underexplored. We aimed to investigate the function of its zebrafish (Danio rerio) ortholog, the dnajb6b gene, in cardiomyopathy and heart failure. MATERIALS AND METHODS Both loss-of-function mutation and gain-of-function transgenic approaches were employed in zebrafish. High frequency echocardiography was performed to evaluate cardiac function indices in adult zebrafish. 4-phenylbutyric acid (4-PBA) was used to pharmacologically inhibit sarcoplasmic reticulum (SR) stress in zebrafish. Western blot was carried out to determine expression of DNAJB6 isoforms in human patients' heart tissues. KEY FINDINGS Global loss-of-function mutations affecting both the sarcomere-localized short (Dnajb6b[S]) and nucleus-localized long (Dnajb6b[L]) isoforms appeared phenotypically normal. In contrast, cardiomyocyte-specific overexpression of a truncated, sarcomere-localized Dnajb6b(L) isoform (Dnajb6b[∆L]) led to severe cardiomyopathy and heart failure phenotypes. Mechanistically, Dnajb6b responded to sarcoplasmic reticulum (SR) stress and activation of Dnajb6b(∆L) resulted in elevated SR stress, accumulation of ubiquitinated protein aggregation, and aberrant activation of autophagy. 4-PBA treatment partially rescued cardiac dysfunction and extended the lifespan of zebrafish with cardiomyocyte-specific activation of Dnajb6b(∆L). Finally, elevated expression of both DNAJB6(S) and DNAJB6(L) isoforms was detected in failing human hearts, supporting their clinical relevance. SIGNIFICANCE Gain-of-function mutation in Dnajb6b(∆L) isoform causes cardiomyopathy and heart failure, likely mediated by elevated SR stress. This study enhances our understanding of Dnajb6's role in cardiac proteostasis and highlights its potential as a therapeutic target for the treatment of cardiomyopathy and heart failure.
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Affiliation(s)
- Yuting Liu
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Yajie Jiang
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Taiwei Ma
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Wenjing Dong
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Peng Yang
- Cardiovascular Surgery Department, Second Xiangya Hospital, Central South University, Changsha 410011, China
| | - Lixia Peng
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Baokun Wang
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Chuanhong Wu
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Zhiqiang Li
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Hong Zhang
- Cardiovascular Surgery Department, Second Xiangya Hospital, Central South University, Changsha 410011, China
| | - Yuanchao Sun
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Yujuan Niu
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China
| | - Yonghe Ding
- The Affiliated Hospital of Qingdao University & Biomedical Sciences Institute, Qingdao Medical College of Qingdao University, Qingdao 266021, China.
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Nasim Z, Karim N, Blilou I, Ahn JH. NMD-mediated posttranscriptional regulation fine-tunes the NLR-WRKY regulatory module to modulate bacterial defense response. PLANT SCIENCE : AN INTERNATIONAL JOURNAL OF EXPERIMENTAL PLANT BIOLOGY 2025; 356:112528. [PMID: 40294849 DOI: 10.1016/j.plantsci.2025.112528] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/23/2025] [Revised: 04/08/2025] [Accepted: 04/22/2025] [Indexed: 04/30/2025]
Abstract
Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance system that maintains transcriptome integrity by degrading aberrant RNA transcripts. NMD ensures proper growth and development by preventing autoimmunity through the direct regulation of nucleotide-binding, leucine-rich repeat (NLR) genes. Whether NMD directly regulates WRKY genes remains unclear, despite their upregulation in NMD-deficient plants, and potential feedback between NLRs and WRKYs is also poorly understood. In this study, we showed that NMD also directly regulates a subset of WRKY (WRKY15, 18, 25, 33, 46, 60, and 70) genes, particularly at lower temperatures (16°C). NMD signature-containing transcripts of WRKY46 and WRKY70, selected as representative NMD-regulated WRKY genes, showed increased half-lives in NMD-deficient mutants. Transcriptome analyses showed that these seven NMD-regulated WRKY genes are induced in response to bacterial infection. Potential homologues of these seven NMD-regulated WRKY genes in maize and rice showed similar induction in response to bacterial pathogen infection. Furthermore, these NMD-regulated WRKY genes are induced in plants overexpressing RESISTANT TO P. SYRINGAE 4 (RPS4) in a temperature-dependent manner. By using ChIP-seq and DAP-seq data of WRKY transcription factors, we showed that WRKYs potentially regulate a significant number of NLR genes by directly binding to the W-box in their promoter regions. Taken together, our findings revealed that in addition to the NLRs, the NMD machinery also regulates WRKY genes to keep the basal defense levels in check and the WRKY transcription factors directly regulate NLR genes to constitutes a positive feedback regulatory loop to optimize the plant response to invading pathogens.
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Affiliation(s)
- Zeeshan Nasim
- Department of Molecular Life Sciences, Korea University, Seoul 02841, Republic of Korea.
| | - Nouroz Karim
- Department of Molecular Life Sciences, Korea University, Seoul 02841, Republic of Korea
| | - Ikram Blilou
- Plant Science Program, Biological and Environmental Science and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
| | - Ji Hoon Ahn
- Department of Molecular Life Sciences, Korea University, Seoul 02841, Republic of Korea.
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Kalra S, Coolon JD. Decoding RAP1 's Role in Yeast mRNA Splicing. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.04.04.647307. [PMID: 40291741 PMCID: PMC12026737 DOI: 10.1101/2025.04.04.647307] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/30/2025]
Abstract
Messenger RNA (mRNA) splicing is a fundamental and tightly regulated process in eukaryotes, where the spliceosome removes non-coding sequences from pre-mRNA to produce mature mRNA for protein translation. Alternative splicing enables the generation of multiple RNA isoforms and protein products from a single gene, regulating both isoform diversity and abundance. While splicing is widespread in eukaryotes, only ∼3% of genes in Saccharomyces cerevisiae undergo splicing, with most containing a single intron. However, intron-containing genes, primarily ribosomal protein genes, are highly expressed and constitute about one-third of the total mRNA pool. These genes are transcriptionally regulated by Repressor Activator Protein 1 ( RAP1 ), prompting us to investigate whether RAP1 influences mRNA splicing. Using RNA sequencing, we identified a novel role for RAP1 in alternative splicing, particularly in intron retention (IR) while minor effects were observed on alternative 3' and 5' splice site usage. Many IR-containing transcripts introduced premature termination codons, likely leading to degradation via nonsense-mediated decay (NMD). Consistent with previous literature, genes with predicted NMD in our study also had reduced overall expression levels suggesting that RAP1 plays an important role in this understudied mechanism of gene expression regulation.
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Lai S, Shiraishi H, Sebastian WA, Shimizu N, Umeda R, Ikeuchi M, Kiyota K, Takeno T, Miyazaki S, Yano S, Shimada T, Yoshimura A, Hanada R, Hanada T. Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish. Commun Biol 2024; 7:654. [PMID: 38806677 PMCID: PMC11133409 DOI: 10.1038/s42003-024-06356-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Accepted: 05/20/2024] [Indexed: 05/30/2024] Open
Abstract
SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts. Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS). However, the molecular mechanism underlying HBMS remains unclear. We generated smg9 mutant zebrafish (smg9oi7/oi7) that have a lifespan of approximately 6 months or longer, allowing for analysis of the in vivo function of Smg9 in adults in more detail. smg9oi7/oi7 zebrafish display congenital brain abnormalities and reduced cardiac contraction. Additionally, smg9oi7/oi7 zebrafish exhibit a premature aging phenotype. Analysis of NMD target mRNAs shows a trend toward increased mRNA levels in smg9oi7/oi7 zebrafish. Spermidine oxidase (Smox) is increased in smg9oi7/oi7 zebrafish, resulting in the accumulation of byproducts, reactive oxygen species, and acrolein. The accumulation of smox mRNA due to NMD dysregulation caused by Smg9 deficiency leads to increased oxidative stress, resulting in premature aging.
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Affiliation(s)
- Shaohong Lai
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Hiroshi Shiraishi
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | | | - Nobuyuki Shimizu
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Ryohei Umeda
- Department of Neurophysiology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Mayo Ikeuchi
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Kyoko Kiyota
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Takashi Takeno
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Shuya Miyazaki
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Shinji Yano
- Institute for Research Management, Oita University, Yufu, Oita, Japan
| | - Tatsuo Shimada
- Oita Medical Technology School, Japan College of Judo-Therapy, Acupuncture & Moxibustion Therapy, Oita, Japan
| | - Akihiko Yoshimura
- Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan
| | - Reiko Hanada
- Department of Neurophysiology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Toshikatsu Hanada
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan.
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Alanazi AR, Parkinson GN, Haider S. Structural Motifs at the Telomeres and Their Role in Regulatory Pathways. Biochemistry 2024; 63:827-842. [PMID: 38481135 PMCID: PMC10993422 DOI: 10.1021/acs.biochem.4c00023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2024] [Revised: 02/28/2024] [Accepted: 02/29/2024] [Indexed: 04/04/2024]
Abstract
Telomeres are specialized structures, found at the ends of linear chromosomes in eukaryotic cells, that play a crucial role in maintaining the stability and integrity of genomes. They are composed of repetitive DNA sequences, ssDNA overhangs, and several associated proteins. The length of telomeres is linked to cellular aging in humans, and deficiencies in their maintenance are associated with various diseases. Key structural motifs at the telomeres serve to protect vulnerable chromosomal ends. Telomeric DNA also has the ability to form diverse complex DNA higher-order structures, including T-loops, D-loops, R-loops, G-loops, G-quadruplexes, and i-motifs, in the complementary C-rich strand. While many essential proteins at telomeres have been identified, the intricacies of their interactions and structural details are still not fully understood. This Perspective highlights recent advancements in comprehending the structures associated with human telomeres. It emphasizes the significance of telomeres, explores various telomeric structural motifs, and delves into the structural biology surrounding telomeres and telomerase. Furthermore, telomeric loops, their topologies, and the associated proteins that contribute to the safeguarding of telomeres are discussed.
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Affiliation(s)
- Abeer
F R Alanazi
- UCL
School of Pharmacy, University College London, London WC1N 1AX, United Kingdom
| | - Gary N Parkinson
- UCL
School of Pharmacy, University College London, London WC1N 1AX, United Kingdom
| | - Shozeb Haider
- UCL
School of Pharmacy, University College London, London WC1N 1AX, United Kingdom
- UCL
Centre for Advanced Research Computing, University College London, London WC1H 9RN, United
Kingdom
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6
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Liu Z, Jing C. Two- and three-dimensional sonographic findings of harlequin ichthyosis: case report and literature review. An Bras Dermatol 2023; 98:806-813. [PMID: 37355352 PMCID: PMC10589490 DOI: 10.1016/j.abd.2022.09.013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2022] [Revised: 09/24/2022] [Accepted: 09/30/2022] [Indexed: 06/26/2023] Open
Abstract
BACKGROUND Harlequin ichthyosis (HI) is a rare skin disorder with extremely high lethality due to a mutation of the ABCA12 gene. Because of its rarity and the often-late onset, prenatal screening for HI is extremely difficult, and most pregnant women might easily miss the period for optimal examinations. OBJECTIVE To summarize the sonographic features of HI for prenatal diagnostic purposes. METHODS The authors describe a case of HI with no family history who was diagnosed by using prenatal ultrasound scanning. The sonographic features of HI and the clinical characteristics of pregnant women were summarized by searching relevant literature over nearly two decades. RESULTS The unique sonographic presentations including peeling skin, clenched hands and clubfeet, ectropion, flat nose, fetal growth impairment, polyhydramnios and echogenic amniotic fluid may be primarily related to skin disorders in HI fetuses. The authors also identified a novel pathogenic ABCA12 gene mutation and explained the possible pathogenic mechanisms. STUDY LIMITATIONS Caution should be exercised in summarizing disease characteristics because of the small number of cases, and the authors are faced with the possibility of incomplete case searching. CONCLUSIONS HI has relatively unique sonographic features. Therefore, 2D-ultrasound combined with 3D-ultrasound may be an effective method for the prenatal diagnosis of HI. Moreover, a novel pathogenic ABCA12 gene mutation may provide important clues for future research on the etiology of HI. However, the authors consider that additional studies are needed to provide more evidence for prenatal diagnosis.
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Affiliation(s)
- Zesi Liu
- Department of Gynecology and Obstetrics, The First Affiliated Hospital Dalian Medical University, Dalian, China
| | - Chunli Jing
- Department of Ultrasound of Gynecology and Obstetrics, The Second Affiliated Hospital Dalian Medical University, Dalian, China.
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Ayyildiz D, Bergonzoni G, Monziani A, Tripathi T, Döring J, Kerschbamer E, Di Leva F, Pennati E, Donini L, Kovalenko M, Zasso J, Conti L, Wheeler VC, Dieterich C, Piazza S, Dassi E, Biagioli M. CAG repeat expansion in the Huntington's disease gene shapes linear and circular RNAs biogenesis. PLoS Genet 2023; 19:e1010988. [PMID: 37831730 PMCID: PMC10617732 DOI: 10.1371/journal.pgen.1010988] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2022] [Revised: 10/31/2023] [Accepted: 09/19/2023] [Indexed: 10/15/2023] Open
Abstract
Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.
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Affiliation(s)
- Dilara Ayyildiz
- Bioinformatic facility, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
- Biomedical Sciences and Biotechnology, University of Udine, Udine, Italy
| | - Guendalina Bergonzoni
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Alan Monziani
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Takshashila Tripathi
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Jessica Döring
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Emanuela Kerschbamer
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Francesca Di Leva
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Elia Pennati
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Luisa Donini
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Marina Kovalenko
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America
| | - Jacopo Zasso
- Laboratory of Stem Cell Biology, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Luciano Conti
- Laboratory of Stem Cell Biology, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Vanessa C. Wheeler
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Department of Neurology Harvard Medical School, Boston, Massachusetts, United States of America
| | - Christoph Dieterich
- Section of Bioinformatics and Systems Cardiology, University Hospital Heidelberg, Heidelberg, Germany
| | - Silvano Piazza
- Bioinformatic facility, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Erik Dassi
- Laboratory of RNA Regulatory Networks, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
| | - Marta Biagioli
- NeuroEpigenetics laboratory, Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy
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Cho S, Chun Y, He L, Ramirez CB, Ganesh KS, Jeong K, Song J, Cheong JG, Li Z, Choi J, Kim J, Koundouros N, Ding F, Dephoure N, Jang C, Blenis J, Lee G. FAM120A couples SREBP-dependent transcription and splicing of lipogenesis enzymes downstream of mTORC1. Mol Cell 2023; 83:3010-3026.e8. [PMID: 37595559 PMCID: PMC10494788 DOI: 10.1016/j.molcel.2023.07.017] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Revised: 05/23/2023] [Accepted: 07/15/2023] [Indexed: 08/20/2023]
Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.
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Affiliation(s)
- Sungyun Cho
- Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Yujin Chun
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, CA, USA
| | - Long He
- Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Cuauhtemoc B Ramirez
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, CA, USA; Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA, USA
| | - Kripa S Ganesh
- Department of Biochemistry, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Kyungjo Jeong
- Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, South Korea
| | - Junho Song
- Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, South Korea
| | - Jin Gyu Cheong
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Zhongchi Li
- Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Jungmin Choi
- Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, South Korea; Department of Genetics, Yale School of Medicine, Yale University, New Haven, CT, USA
| | - Joohwan Kim
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, CA, USA
| | - Nikos Koundouros
- Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY, USA; Meyer Cancer Center, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Fangyuan Ding
- Department of Biomedical Engineering, Department of Developmental and Cell Biology, Department of Pharmaceutical Sciences, Center for Synthetic Biology, and Center for Neural Circuit Mapping, University of California Irvine, Irvine, CA, USA; Center for Complex Biological Systems and Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, USA
| | - Noah Dephoure
- Meyer Cancer Center, Weill Cornell Medicine, Cornell University, New York, NY, USA
| | - Cholsoon Jang
- Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA, USA; Center for Complex Biological Systems and Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, USA; Center for Epigenetics and Metabolism, University of California Irvine, Irvine, CA, USA
| | - John Blenis
- Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY, USA; Meyer Cancer Center, Weill Cornell Medicine, Cornell University, New York, NY, USA.
| | - Gina Lee
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, CA, USA; Center for Complex Biological Systems and Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, USA; Center for Epigenetics and Metabolism, University of California Irvine, Irvine, CA, USA.
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9
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Becker JT, Auerbach AA, Harris RS. APEX3 - an optimized tool for rapid and unbiased proximity labeling. J Mol Biol 2023; 435:168145. [PMID: 37182813 DOI: 10.1016/j.jmb.2023.168145] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Revised: 05/03/2023] [Accepted: 05/09/2023] [Indexed: 05/16/2023]
Abstract
Macromolecular interactions regulate all aspects of biology. The identification of interacting partners and complexes is important for understanding cellular processes, host-pathogen conflicts, and organismal development. Multiple methods exist to label and enrich interacting proteins in living cells. Notably, the soybean ascorbate peroxidase, APEX2, rapidly biotinylates adjacent biomolecules in the presence of biotin-phenol and hydrogen peroxide. However, during initial experiments with this system, we found that APEX2 exhibits a cytoplasmic-biased localization and is sensitive to the nuclear export inhibitor leptomycin B (LMB). This led us to identify a putative nuclear export signal (NES) at the carboxy-terminus of APEX2 (NESAPEX2), structurally adjacent to the conserved heme binding site. This putative NES is functional as evidenced by cytoplasmic localization and LMB sensitivity of a mCherry-NESAPEX2 chimeric construct. Single amino acid substitutions of multiple hydrophobic residues within NESAPEX2 eliminate cytoplasm-biased localization of both mCherry-NESAPEX2 as well as full-length APEX2. However, all but one of these NES substitutions also compromises peroxide-dependent labeling. This unique separation-of-function mutant, APEX2-L242A, is termed APEX3. Localization and functionality of APEX3 are confirmed by fusion to the nucleocytoplasmic shuttling transcriptional factor, RELA. APEX3 is therefore an optimized tool for unbiased proximity labeling of cellular proteins and interacting factors.
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Affiliation(s)
- Jordan T Becker
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN, USA 55455; Department of Microbiology and Immunology, University of Minnesota Twin Cities, Minneapolis, MN, USA 55455; Institute for Molecular Virology, University of Minnesota Twin Cities, Minneapolis, MN, USA 55455.
| | - Ashley A Auerbach
- Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX, USA 78229
| | - Reuben S Harris
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN, USA 55455; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX, USA 78229; Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, TX, USA 78229.
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Wagner RN, Wießner M, Friedrich A, Zandanell J, Breitenbach-Koller H, Bauer JW. Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond. Int J Mol Sci 2023; 24:6101. [PMID: 37047074 PMCID: PMC10093890 DOI: 10.3390/ijms24076101] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Revised: 03/17/2023] [Accepted: 03/21/2023] [Indexed: 04/14/2023] Open
Abstract
Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.
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Affiliation(s)
- Roland N. Wagner
- Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria
| | - Michael Wießner
- Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria
| | - Andreas Friedrich
- Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria
- Department of Biosciences, University of Salzburg, 5020 Salzburg, Austria
| | - Johanna Zandanell
- Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria
| | | | - Johann W. Bauer
- Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria
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11
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V M DD, Sivaramakrishnan V, Arvind Kumar K. Structural systems biology approach delineate the functional implications of SNPs in exon junction complex interaction network. J Biomol Struct Dyn 2023; 41:11969-11986. [PMID: 36617892 DOI: 10.1080/07391102.2022.2164355] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 12/26/2022] [Indexed: 01/10/2023]
Abstract
In eukaryotes, transcripts that carry premature termination codons (PTC) leading to truncated proteins are degraded by the Nonsense Mediated Decay (NMD) machinery. Missense and nonsense Single Nucleotide Polymorphisms (SNPs) in proteins belonging to Exon junction complex (EJC) and up-frameshift protein (UPF) will compromise NMD leading to the accumulation of truncated proteins in various diseases. The EJC and UPF which are involved in NMD is a good model system to study the effect of SNPs at a system level. Despite the availability of crystal structures, computational tools, and data on mutational and deletion studies, with functional implications, an integrated effort to understand the impact of SNPs at the systems level is lacking. To study the functional consequences of missense SNPs, sequence-based techniques like SIFT and PolyPhen which classify SNPs as deleterious or non-deleterious and structure-based methods like FoldX which calculate the Delta Delta G, (ddGs, ∆∆G) are used. Using FoldX, the ddG for mutations with experimentally validated functional effects is calculated and compared with those calculated for SNPs in the same protein-protein interaction interface. Further, a model is conceived to explain the functional implications of SNPs based on the effects observed for known mutants. The results are visualized in a network format. The effects of nonsense mutations are discerned by comparing with deletion mutation studies and loss of interaction in the crystal structure. The present work not only integrates genomics, proteomics, and classical genetics with 'Structural Biology' but also helps to integrate it into a 'systems-level functional network'.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Datta Darshan V M
- Disease Biology Lab, Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Anantapur, Andhra Pradesh, India
| | - Venketesh Sivaramakrishnan
- Disease Biology Lab, Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Anantapur, Andhra Pradesh, India
| | - K Arvind Kumar
- Disease Biology Lab, Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Anantapur, Andhra Pradesh, India
- Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH, USA
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12
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Zhou Z, Hu F, Huang D, Chi Q, Tang NLS. Nonsense-Mediated Decay Targeted RNA (ntRNA): Proposal of a ntRNA–miRNA–lncRNA Triple Regulatory Network Usable as Biomarker of Prognostic Risk in Patients with Kidney Cancer. Genes (Basel) 2022; 13:genes13091656. [PMID: 36140823 PMCID: PMC9498815 DOI: 10.3390/genes13091656] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 09/05/2022] [Accepted: 09/08/2022] [Indexed: 11/16/2022] Open
Abstract
The most prevalent subtype of renal cell carcinoma (RCC), kidney renal clear cell carcinoma (KIRC) may be associated with a poor prognosis in a high number of cases, with a stage-specific prognostic stratification currently in use. No reliable biomarkers have been utilized so far in clinical practice despite the efforts in biomarker research in the last years. Nonsense-mediated mRNA decay (NMD) is a critical safeguard against erroneous transcripts, particularly mRNA transcripts containing premature termination codons (called nonsense-mediated decay targeted RNA, ntRNA). In this study, we first characterized 296 differentially expressed ntRNAs that were independent of the corresponding gene, 261 differentially expressed miRNAs, and 4653 differentially expressed lncRNAs. Then, we constructed a hub ntRNA–miRNA–lncRNA triple regulatory network associated with the prognosis of KIRC. Moreover, the results of immune infiltration analysis indicated that this network may influence the changes of the tumor immune microenvironment. A prognostic model derived from the genes and immune cells associated with the network was developed to distinguish between high- and low-risk patients, which was a better prognostic than other models, constructed using different biomarkers. Additionally, correlation of methylation and ntRNAs in the network suggested that some ntRNAs were regulated by methylation, which is helpful to further study the causes of abnormal expression of ntRNAs. In conclusion, this study highlighted the possible clinical implications of ntRNA functions in KIRC, proposing potential significant biomarkers that could be utilized to define the prognosis and design personalized treatment plans in kidney cancer management in the next future.
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Affiliation(s)
- Zhiyue Zhou
- Department of Statistics, School of Science, Wuhan University of Technology, 122 Luoshi Road, Wuhan 430070, China
| | - Fuyan Hu
- Department of Statistics, School of Science, Wuhan University of Technology, 122 Luoshi Road, Wuhan 430070, China
- Correspondence: (F.H.); (N.L.S.T.)
| | - Dan Huang
- Department of Biology, Southern University of Science and Technology, Shenzhen 518055, China
| | - Qingjia Chi
- Department of Engineering Structure and Mechanics, School of Science, Wuhan University of Technology, Wuhan 430070, China
| | - Nelson L. S. Tang
- Department of Chemical Pathology and Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
- Functional Genomics and Biostatistical Computing Laboratory, CUHK Shenzhen Research Institute, Shenzhen 518000, China
- Hong Kong Branch of CAS Center for Excellence in Animal Evolution and Genetics, School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China
- Correspondence: (F.H.); (N.L.S.T.)
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13
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Martin H, Rupkey J, Asthana S, Yoon J, Patel S, Mott J, Pei Z, Mao Y. Diverse Roles of the Exon Junction Complex Factors in the Cell Cycle, Cancer, and Neurodevelopmental Disorders-Potential for Therapeutic Targeting. Int J Mol Sci 2022; 23:ijms231810375. [PMID: 36142288 PMCID: PMC9499366 DOI: 10.3390/ijms231810375] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 09/01/2022] [Accepted: 09/05/2022] [Indexed: 12/04/2022] Open
Abstract
The exon junction complex (EJC) plays a crucial role in regulating gene expression at the levels of alternative splicing, translation, mRNA localization, and nonsense-mediated decay (NMD). The EJC is comprised of three core proteins: RNA-binding motif 8A (RBM8A), Mago homolog (MAGOH), eukaryotic initiation factor 4A3 (eIF4A3), and a peripheral EJC factor, metastatic lymph node 51 (MLN51), in addition to other peripheral factors whose structural integration is activity-dependent. The physiological and mechanistic roles of the EJC in contribution to molecular, cellular, and organismal level function continue to be explored for potential insights into genetic or pathological dysfunction. The EJC’s specific role in the cell cycle and its implications in cancer and neurodevelopmental disorders prompt enhanced investigation of the EJC as a potential target for these diseases. In this review, we highlight the current understanding of the EJC’s position in the cell cycle, its relation to cancer and developmental diseases, and potential avenues for therapeutic targeting.
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Affiliation(s)
- Hannah Martin
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
| | - Julian Rupkey
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
| | - Shravan Asthana
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
- Feinberg School of Medicine, Northwestern University, 303 East Superior Street, Chicago, IL 60611, USA
| | - Joy Yoon
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
| | - Shray Patel
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
| | - Jennifer Mott
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
| | - Zifei Pei
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
| | - Yingwei Mao
- Department of Biology, Pennsylvania State University, University Park, State College, PA 16802, USA
- Correspondence:
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14
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Pan YJ, Liu BW, Pei DS. The Role of Alternative Splicing in Cancer: Regulatory Mechanism, Therapeutic Strategy, and Bioinformatics Application. DNA Cell Biol 2022; 41:790-809. [PMID: 35947859 DOI: 10.1089/dna.2022.0322] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
[Formula: see text] Alternative splicing (AS) can generate distinct transcripts and subsequent isoforms that play differential functions from the same pre-mRNA. Recently, increasing numbers of studies have emerged, unmasking the association between AS and cancer. In this review, we arranged AS events that are closely related to cancer progression and presented promising treatments based on AS for cancer therapy. Obtaining proliferative capacity, acquiring invasive properties, gaining angiogenic features, shifting metabolic ability, and getting immune escape inclination are all splicing events involved in biological processes. Spliceosome-targeted and antisense oligonucleotide technologies are two novel strategies that are hopeful in tumor therapy. In addition, bioinformatics applications based on AS were summarized for better prediction and elucidation of regulatory routines mingled in. Together, we aimed to provide a better understanding of complicated AS events associated with cancer biology and reveal AS a promising target of cancer treatment in the future.
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Affiliation(s)
- Yao-Jie Pan
- Department of Pathology, Laboratory of Clinical and Experimental Pathology, Xuzhou Medical University, Xuzhou, China
| | - Bo-Wen Liu
- Department of General Surgery, Xuzhou Medical University, Xuzhou, China
| | - Dong-Sheng Pei
- Department of Pathology, Laboratory of Clinical and Experimental Pathology, Xuzhou Medical University, Xuzhou, China
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15
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Cabezas-Fuster A, Micol-Ponce R, Fontcuberta-Cervera S, Ponce M. Missplicing suppressor alleles of Arabidopsis PRE-MRNA PROCESSING FACTOR 8 increase splicing fidelity by reducing the use of novel splice sites. Nucleic Acids Res 2022; 50:5513-5527. [PMID: 35639749 PMCID: PMC9177961 DOI: 10.1093/nar/gkac338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Revised: 03/30/2022] [Accepted: 04/25/2022] [Indexed: 11/21/2022] Open
Abstract
Efficient splicing requires a balance between high-fidelity splice-site (SS) selection and speed. In Saccharomyces cerevisiae, Pre-mRNA processing factor 8 (Prp8) helps to balance precise SS selection and rapid, efficient intron excision and exon joining. argonaute1-52 (ago1-52) and incurvata13 (icu13) are hypomorphic alleles of the Arabidopsis thaliana genes ARGONAUTE1 (AGO1) and AUXIN RESISTANT6 (AXR6) that harbor point mutations creating a novel 3'SS and 5'SS, respectively. The spliceosome recognizes these novel SSs, as well as the intact genuine SSs, producing a mixture of wild-type and aberrant mature mRNAs. Here, we characterized five novel mutant alleles of PRP8 (one of the two Arabidopsis co-orthologs of yeast Prp8), naming these alleles morphology of ago1-52 suppressed5 (mas5). In the mas5-1 background, the spliceosome preferentially recognizes the intact genuine 3'SS of ago1-52 and 5'SS of icu13. Since point mutations that damage genuine SSs make the spliceosome prone to recognizing cryptic SSs, we also tested alleles of four genes carrying damaged genuine SSs, finding that mas5-1 did not suppress their missplicing. The mas5-1 and mas5-3 mutations represent a novel class of missplicing suppressors that increase splicing fidelity by hampering the use of novel SSs, but do not alter general pre-mRNA splicing.
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Affiliation(s)
- Adrián Cabezas-Fuster
- Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante, Spain
| | - Rosa Micol-Ponce
- Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante, Spain
| | - Sara Fontcuberta-Cervera
- Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante, Spain
| | - María Rosa Ponce
- Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante, Spain
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16
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Richter F, Plehn JE, Bessler L, Hertler J, Jörg M, Cirzi C, Tuorto F, Friedland K, Helm M. RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis. Nucleic Acids Res 2022; 50:4201-4215. [PMID: 34850949 PMCID: PMC9071408 DOI: 10.1093/nar/gkab1150] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Revised: 10/31/2021] [Accepted: 11/03/2021] [Indexed: 12/16/2022] Open
Abstract
The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.
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Affiliation(s)
- Florian Richter
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany
| | - Johanna E Plehn
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany
| | - Larissa Bessler
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany
| | - Jasmin Hertler
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany
| | - Marko Jörg
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany
| | - Cansu Cirzi
- Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ) 69120 Heidelberg, Germany
- Faculty of Biosciences, University of Heidelberg 69120 Heidelberg, Germany
| | - Francesca Tuorto
- Division of Biochemistry, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
- Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance69120 Heidelberg, Germany
| | - Kristina Friedland
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany
| | - Mark Helm
- Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany
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17
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Mesa-Perez M, Hamilton PT, Miranda A, Brodie N, O’Sullivan C, Christie J, Ryan B, Chow R, Goodlett D, Nelson C, Howard P. Cytoplasmic switch of ARS2 isoforms promotes nonsense-mediated mRNA decay and arsenic sensitivity. Nucleic Acids Res 2022; 50:1620-1638. [PMID: 35104878 PMCID: PMC8860587 DOI: 10.1093/nar/gkac033] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 12/29/2021] [Accepted: 01/13/2022] [Indexed: 12/03/2022] Open
Abstract
The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), bound to the cap binding complex (CBC). We report here that alternative splicing of ARS2's intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to NCBP1 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress, where cytoplasmic ARS2 promotes a global response to arsenic in a CBC-independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, and stress response.
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Affiliation(s)
- Monica Mesa-Perez
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada
| | | | - Alex Miranda
- Deeley Research Centre, BC Cancer, Victoria, BC V8R 6V5, Canada
| | - Nicholas Brodie
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada
- University of Victoria Genome BC Proteomics Centre, Vancouver Island Technology Park, Victoria, BC V8Z 7X8, Canada
| | - Connor O’Sullivan
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada
| | - Jennifer Christie
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada
| | - Bridget C Ryan
- Department of Biology, University of Victoria, Victoria, BC V8W 3N5, Canada
| | - Robert L Chow
- Department of Biology, University of Victoria, Victoria, BC V8W 3N5, Canada
| | - David Goodlett
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada
- University of Victoria Genome BC Proteomics Centre, Vancouver Island Technology Park, Victoria, BC V8Z 7X8, Canada
| | - Christopher J Nelson
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada
| | - Perry L Howard
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada
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18
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Nasim Z, Fahim M, Hwang H, Susila H, Jin S, Youn G, Ahn JH. Nonsense-mediated mRNA decay modulates Arabidopsis flowering time via the SET DOMAIN GROUP 40-FLOWERING LOCUS C module. JOURNAL OF EXPERIMENTAL BOTANY 2021; 72:7049-7066. [PMID: 34270724 DOI: 10.1093/jxb/erab331] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Accepted: 07/14/2021] [Indexed: 06/13/2023]
Abstract
The nonsense-mediated mRNA decay (NMD) surveillance system clears aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. Although loss of the core NMD proteins UP-FRAMESHIFT1 (UPF1) and UPF3 leads to late flowering in Arabidopsis, the underlying mechanism remains elusive. Here, we showed that mutations in UPF1 and UPF3 cause temperature- and photoperiod-independent late flowering. Expression analyses revealed high FLOWERING LOCUS C (FLC) mRNA levels in upf mutants; in agreement with this, the flc mutation strongly suppressed the late flowering of upf mutants. Vernalization accelerated flowering of upf mutants in a temperature-independent manner. FLC transcript levels rose in wild-type plants upon NMD inhibition. In upf mutants, we observed increased enrichment of H3K4me3 and reduced enrichment of H3K27me3 in FLC chromatin. Transcriptome analyses showed that SET DOMAIN GROUP 40 (SDG40) mRNA levels increased in upf mutants, and the SDG40 transcript underwent NMD-coupled alternative splicing, suggesting that SDG40 affects flowering time in upf mutants. Furthermore, NMD directly regulated SDG40 transcript stability. The sdg40 mutants showed decreased H3K4me3 and increased H3K27me3 levels in FLC chromatin, flowered early, and rescued the late flowering of upf mutants. Taken together, these results suggest that NMD epigenetically regulates FLC through SDG40 to modulate flowering time in Arabidopsis.
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Affiliation(s)
- Zeeshan Nasim
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Muhammad Fahim
- Centre for Omic Sciences, Islamia College Peshawar, Pakistan
| | - Hocheol Hwang
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Hendry Susila
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Suhyun Jin
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Geummin Youn
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Ji Hoon Ahn
- Department of Life Sciences, Korea University, Seoul 02841, Korea
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19
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Ma T, Gao H, Zhang D, Sun W, Yin Q, Wu L, Zhang T, Xu Z, Wei J, Su Y, Shi Y, Ding D, Yuan L, Dong G, Leng L, Xiang L, Chen S. Genome-Wide Analysis of Light-Regulated Alternative Splicing in Artemisia annua L. FRONTIERS IN PLANT SCIENCE 2021; 12:733505. [PMID: 34659300 PMCID: PMC8511310 DOI: 10.3389/fpls.2021.733505] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/06/2021] [Accepted: 09/03/2021] [Indexed: 06/13/2023]
Abstract
Artemisinin is currently the most effective ingredient in the treatment of malaria, which is thus of great significance to study the genetic regulation of Artemisia annua. Alternative splicing (AS) is a regulatory process that increases the complexity of transcriptome and proteome. The most common mechanism of alternative splicing (AS) in plant is intron retention (IR). However, little is known about whether the IR isoforms produced by light play roles in regulating biosynthetic pathways. In this work we would explore how the level of AS in A. annua responds to light regulation. We obtained a new dataset of AS by analyzing full-length transcripts using both Illumina- and single molecule real-time (SMRT)-based RNA-seq as well as analyzing AS on various tissues. A total of 5,854 IR isoforms were identified, with IR accounting for the highest proportion (48.48%), affirming that IR is the most common mechanism of AS. We found that the number of up-regulated IR isoforms (1534/1378, blue and red light, respectively) was more than twice that of down-regulated (636/682) after treatment of blue or red light. In the artemisinin biosynthetic pathway, 10 genes produced 16 differentially expressed IR isoforms. This work demonstrated that the differential expression of IR isoforms induced by light has the potential to regulate sesquiterpenoid biosynthesis. This study also provides high accuracy full-length transcripts, which can be a valuable genetic resource for further research of A. annua, including areas of development, breeding, and biosynthesis of active compounds.
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Affiliation(s)
- Tingyu Ma
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
- Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People’s Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Han Gao
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
- School of Life Sciences, Central China Normal University, Wuhan, China
| | - Dong Zhang
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
- College of Agriculture, South China Agricultural University, Guangzhou, China
- Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China
| | - Wei Sun
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
| | - Qinggang Yin
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
| | - Lan Wu
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
| | - Tianyuan Zhang
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China
| | - Zhichao Xu
- Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People’s Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Jianhe Wei
- Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou, China
| | - Yanyan Su
- Amway (China) Botanical R&D Center, Wuxi, China
| | - Yuhua Shi
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
| | - Dandan Ding
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
| | - Ling Yuan
- Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, United States
| | | | - Liang Leng
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
| | - Li Xiang
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
- Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, United States
| | - Shilin Chen
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
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20
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Olthof AM, White AK, Mieruszynski S, Doggett K, Lee MF, Chakroun A, Abdel Aleem AK, Rousseau J, Magnani C, Roifman CM, Campeau PM, Heath JK, Kanadia RN. Disruption of exon-bridging interactions between the minor and major spliceosomes results in alternative splicing around minor introns. Nucleic Acids Res 2021; 49:3524-3545. [PMID: 33660780 PMCID: PMC8034651 DOI: 10.1093/nar/gkab118] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 02/08/2021] [Accepted: 02/10/2021] [Indexed: 12/11/2022] Open
Abstract
Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry–Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.
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Affiliation(s)
- Anouk M Olthof
- Physiology and Neurobiology Department, University of Connecticut, 75 N. Eagleville Road, Storrs, CT 06269, USA
| | - Alisa K White
- Physiology and Neurobiology Department, University of Connecticut, 75 N. Eagleville Road, Storrs, CT 06269, USA
| | - Stephen Mieruszynski
- Epigenetics and Development Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia
| | - Karen Doggett
- Epigenetics and Development Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia
| | - Madisen F Lee
- Physiology and Neurobiology Department, University of Connecticut, 75 N. Eagleville Road, Storrs, CT 06269, USA
| | | | | | - Justine Rousseau
- CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - Cinzia Magnani
- Neonatology and Neonatal Intensive Care Unit, Maternal and Child Department, University of Parma, Parma, 43121, Italy
| | - Chaim M Roifman
- Division of Immunology and Allergy, Department of Pediatrics, The Hospital for Sick Children and the University of Toronto, Toronto, ON M5G 1X8, Canada.,The Canadian Centre for Primary Immunodeficiency and The Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
| | - Philippe M Campeau
- Department of Pediatrics, University of Montreal, Montreal, QC H4A 3J1, Canada
| | - Joan K Heath
- Epigenetics and Development Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia
| | - Rahul N Kanadia
- Physiology and Neurobiology Department, University of Connecticut, 75 N. Eagleville Road, Storrs, CT 06269, USA.,Institute for System Genomics, University of Connecticut, Storrs, CT 06269, USA
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21
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Oh J, Pradella D, Shao C, Li H, Choi N, Ha J, Ruggiero S, Fu XD, Zheng X, Ghigna C, Shen H. Widespread Alternative Splicing Changes in Metastatic Breast Cancer Cells. Cells 2021; 10:cells10040858. [PMID: 33918758 PMCID: PMC8070448 DOI: 10.3390/cells10040858] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2021] [Revised: 04/05/2021] [Accepted: 04/06/2021] [Indexed: 12/11/2022] Open
Abstract
Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types.
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Affiliation(s)
- Jagyeong Oh
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; (J.O.); (N.C.); (J.H.); (X.Z.)
| | - Davide Pradella
- Institute of Molecular Genetics “Luigi Luca Cavalli-Sforza”, National Research Council, Via Abbiategrasso 207, 27100 Pavia, Italy; (D.P.); (S.R.)
| | - Changwei Shao
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0021, USA; (C.S.); (H.L.); (X.-D.F.)
| | - Hairi Li
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0021, USA; (C.S.); (H.L.); (X.-D.F.)
| | - Namjeong Choi
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; (J.O.); (N.C.); (J.H.); (X.Z.)
| | - Jiyeon Ha
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; (J.O.); (N.C.); (J.H.); (X.Z.)
| | - Sonia Ruggiero
- Institute of Molecular Genetics “Luigi Luca Cavalli-Sforza”, National Research Council, Via Abbiategrasso 207, 27100 Pavia, Italy; (D.P.); (S.R.)
| | - Xiang-Dong Fu
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0021, USA; (C.S.); (H.L.); (X.-D.F.)
| | - Xuexiu Zheng
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; (J.O.); (N.C.); (J.H.); (X.Z.)
| | - Claudia Ghigna
- Institute of Molecular Genetics “Luigi Luca Cavalli-Sforza”, National Research Council, Via Abbiategrasso 207, 27100 Pavia, Italy; (D.P.); (S.R.)
- Correspondence: (C.G.); (H.S.); Tel.: +39-0382-546324 (C.G.); +82-62-715-2507 (H.S.); Fax: +39-0382-422-286 (C.G.); +82-62-715-2484 (H.S.)
| | - Haihong Shen
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; (J.O.); (N.C.); (J.H.); (X.Z.)
- Correspondence: (C.G.); (H.S.); Tel.: +39-0382-546324 (C.G.); +82-62-715-2507 (H.S.); Fax: +39-0382-422-286 (C.G.); +82-62-715-2484 (H.S.)
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22
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Lee PJ, Yang S, Sun Y, Guo JU. Regulation of nonsense-mediated mRNA decay in neural development and disease. J Mol Cell Biol 2021; 13:269-281. [PMID: 33783512 PMCID: PMC8339359 DOI: 10.1093/jmcb/mjab022] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Revised: 01/27/2021] [Accepted: 02/05/2021] [Indexed: 11/26/2022] Open
Abstract
Eukaryotes have evolved a variety of mRNA surveillance mechanisms to detect and degrade aberrant mRNAs with potential deleterious outcomes. Among them, nonsense-mediated mRNA decay (NMD) functions not only as a quality control mechanism targeting aberrant mRNAs containing a premature termination codon but also as a posttranscriptional gene regulation mechanism targeting numerous physiological mRNAs. Despite its well-characterized molecular basis, the regulatory scope and biological functions of NMD at an organismal level are incompletely understood. In humans, mutations in genes encoding core NMD factors cause specific developmental and neurological syndromes, suggesting a critical role of NMD in the central nervous system. Here, we review the accumulating biochemical and genetic evidence on the developmental regulation and physiological functions of NMD as well as an emerging role of NMD dysregulation in neurodegenerative diseases.
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Affiliation(s)
- Paul Jongseo Lee
- Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA.,Interdepartmental Neuroscience Program, Yale University, New Haven, CT 06520, USA
| | - Suzhou Yang
- Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA.,Interdepartmental Neuroscience Program, Yale University, New Haven, CT 06520, USA
| | - Yu Sun
- Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Junjie U Guo
- Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA.,Interdepartmental Neuroscience Program, Yale University, New Haven, CT 06520, USA
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23
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Guo Y, Tocchini C, Ciosk R. CLK-2/TEL2 is a conserved component of the nonsense-mediated mRNA decay pathway. PLoS One 2021; 16:e0244505. [PMID: 33444416 PMCID: PMC7808604 DOI: 10.1371/journal.pone.0244505] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Accepted: 12/10/2020] [Indexed: 11/19/2022] Open
Abstract
Nonsense-mediated mRNA decay (NMD) controls eukaryotic mRNA quality, inducing the degradation of faulty transcripts. Key players in the NMD pathway were originally identified, through genetics, in Caenorhabditis elegans as smg (suppressor with morphological effect on genitalia) genes. Using forward genetics and fluorescence-based NMD reporters, we reexamined the genetic landscape underlying NMD. Employing a novel strategy for mapping sterile mutations, Het-Map, we identified clk-2, a conserved gene previously implicated in DNA damage signaling, as a player in the nematode NMD. We find that CLK-2 is expressed predominantly in the germline, highlighting the importance of auxiliary factors in tissue-specific mRNA decay. Importantly, the human counterpart of CLK-2/TEL2, TELO2, has been also implicated in the NMD, suggesting a conserved role of CLK-2/TEL2 proteins in mRNA surveillance. Recently, variants of TELO2 have been linked to an intellectual disability disorder, the You-Hoover-Fong syndrome, which could be related to its function in the NMD.
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Affiliation(s)
- Yanwu Guo
- Department of Biosciences, University of Oslo, Oslo, Norway
| | | | - Rafal Ciosk
- Department of Biosciences, University of Oslo, Oslo, Norway
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland
- * E-mail:
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24
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Roudko V, Bozkus CC, Orfanelli T, McClain CB, Carr C, O'Donnell T, Chakraborty L, Samstein R, Huang KL, Blank SV, Greenbaum B, Bhardwaj N. Shared Immunogenic Poly-Epitope Frameshift Mutations in Microsatellite Unstable Tumors. Cell 2020; 183:1634-1649.e17. [PMID: 33259803 PMCID: PMC8025604 DOI: 10.1016/j.cell.2020.11.004] [Citation(s) in RCA: 130] [Impact Index Per Article: 26.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2019] [Revised: 06/22/2020] [Accepted: 11/02/2020] [Indexed: 12/20/2022]
Abstract
Microsatellite instability-high (MSI-H) tumors are characterized by high tumor mutation burden and responsiveness to checkpoint blockade. We identified tumor-specific frameshifts encoding multiple epitopes that originated from indel mutations shared among patients with MSI-H endometrial, colorectal, and stomach cancers. Epitopes derived from these shared frameshifts have high population occurrence rates, wide presence in many tumor subclones, and are predicted to bind to the most frequent MHC alleles in MSI-H patient cohorts. Neoantigens arising from these mutations are distinctly unlike self and viral antigens, signifying novel groups of potentially highly immunogenic tumor antigens. We further confirmed the immunogenicity of frameshift peptides in T cell stimulation experiments using blood mononuclear cells isolated from both healthy donors and MSI-H cancer patients. Our study uncovers the widespread occurrence and strong immunogenicity of tumor-specific antigens derived from shared frameshift mutations in MSI-H cancer and Lynch syndrome patients, suitable for the design of common "off-the-shelf" cancer vaccines.
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Affiliation(s)
- Vladimir Roudko
- Department of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA
| | - Cansu Cimen Bozkus
- Department of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA
| | - Theofano Orfanelli
- Department of Obstetrics, Gynecology and Reproductive Science, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA; The Blavatnik Family Women's Health Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Christopher B McClain
- Department of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA
| | - Caitlin Carr
- Department of Obstetrics, Gynecology and Reproductive Science, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA; The Blavatnik Family Women's Health Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Timothy O'Donnell
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA
| | - Lauren Chakraborty
- Department of Biological Sciences, University of Chicago, Chicago, IL, USA
| | - Robert Samstein
- Department of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA
| | - Kuan-Lin Huang
- Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA
| | - Stephanie V Blank
- Department of Obstetrics, Gynecology and Reproductive Science, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA; The Blavatnik Family Women's Health Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Benjamin Greenbaum
- Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Nina Bhardwaj
- Department of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA.
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25
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Zhang Z, Tang J, He X, Di R, Zhang X, Zhang J, Hu W, Chu M. Identification and Characterization of Hypothalamic Alternative Splicing Events and Variants in Ovine Fecundity-Related Genes. Animals (Basel) 2020; 10:ani10112111. [PMID: 33203033 PMCID: PMC7698220 DOI: 10.3390/ani10112111] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2020] [Revised: 11/03/2020] [Accepted: 11/10/2020] [Indexed: 12/03/2022] Open
Abstract
Simple Summary Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction. However, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. In this study, we performed a correlation analysis of transcriptomics and proteomics, and the results suggested several differentially expressed genes (DEGs)/differentially expressed proteins (DEPs), including galectin 3 (LGALS3), aspartoacylase (ASPA) and transthyretin (TTR), could be candidate genes influencing ovine litter size. Further analysis suggested that AS events, single nucleotide polymorphisms (SNPs) and microRNA (miRNA)-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. This study provides a new insight into ovine and even other mammalian reproduction. Abstract Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction; however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small Tail Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as galectin 3 (LGALS3) in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction.
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Affiliation(s)
- Zhuangbiao Zhang
- Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; (Z.Z.); (J.T.); (X.H.); (R.D.)
| | - Jishun Tang
- Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; (Z.Z.); (J.T.); (X.H.); (R.D.)
- Institute of Animal Husbandry and Veterinary Medicine, Anhui Academy of Agricultural Sciences, Hefei 230031, China
| | - Xiaoyun He
- Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; (Z.Z.); (J.T.); (X.H.); (R.D.)
| | - Ran Di
- Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; (Z.Z.); (J.T.); (X.H.); (R.D.)
| | - Xiaosheng Zhang
- Tianjin Institute of Animal Sciences, Tianjin 300381, China; (X.Z.); (J.Z.)
| | - Jinlong Zhang
- Tianjin Institute of Animal Sciences, Tianjin 300381, China; (X.Z.); (J.Z.)
| | - Wenping Hu
- Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; (Z.Z.); (J.T.); (X.H.); (R.D.)
- Correspondence: (W.H.); (M.C.); Tel.: +86-010-6281-6002 (W.H.); +86-010-6281-9850 (M.C.)
| | - Mingxing Chu
- Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; (Z.Z.); (J.T.); (X.H.); (R.D.)
- Correspondence: (W.H.); (M.C.); Tel.: +86-010-6281-6002 (W.H.); +86-010-6281-9850 (M.C.)
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26
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Nasim Z, Fahim M, Gawarecka K, Susila H, Jin S, Youn G, Ahn JH. Role of AT1G72910, AT1G72940, and ADR1-LIKE 2 in Plant Immunity under Nonsense-Mediated mRNA Decay-Compromised Conditions at Low Temperatures. Int J Mol Sci 2020; 21:E7986. [PMID: 33121126 PMCID: PMC7663611 DOI: 10.3390/ijms21217986] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 10/23/2020] [Accepted: 10/23/2020] [Indexed: 01/26/2023] Open
Abstract
Nonsense-mediated mRNA decay (NMD) removes aberrant transcripts to avoid the accumulation of truncated proteins. NMD regulates nucleotide-binding, leucine-rich repeat (NLR) genes to prevent autoimmunity; however, the function of a large number of NLRs still remains poorly understood. Here, we show that three NLR genes (AT1G72910, AT1G72940, and ADR1-LIKE 2) are important for NMD-mediated regulation of defense signaling at lower temperatures. At 16 °C, the NMD-compromised up-frameshift protein1 (upf1) upf3 mutants showed growth arrest that can be rescued by the artificial miRNA-mediated knockdown of the three NLR genes. mRNA levels of these NLRs are induced by Pseudomonas syringae inoculation and exogenous SA treatment. Mutations in AT1G72910, AT1G72940, and ADR1-LIKE 2 genes resulted in increased susceptibility to Pseudomonas syringae, whereas their overexpression resulted in severely stunted growth, which was dependent on basal disease resistance genes. The NMD-deficient upf1 upf3 mutants accumulated higher levels of NMD signature-containing transcripts from these NLR genes at 16 °C. Furthermore, mRNA degradation kinetics showed that these NMD signature-containing transcripts were more stable in upf1 upf3 mutants. Based on these findings, we propose that AT1G72910, AT1G72940, and ADR1-LIKE 2 are directly regulated by NMD in a temperature-dependent manner and play an important role in modulating plant immunity at lower temperatures.
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Affiliation(s)
- Zeeshan Nasim
- Department of Life Sciences, Korea University, Seoul 02841, Korea; (Z.N.); (K.G.); (H.S.); (S.J.); (G.Y.)
| | - Muhammad Fahim
- Centre for Omic Sciences, Islamia College University, Peshawar 25120, Pakistan;
| | - Katarzyna Gawarecka
- Department of Life Sciences, Korea University, Seoul 02841, Korea; (Z.N.); (K.G.); (H.S.); (S.J.); (G.Y.)
| | - Hendry Susila
- Department of Life Sciences, Korea University, Seoul 02841, Korea; (Z.N.); (K.G.); (H.S.); (S.J.); (G.Y.)
| | - Suhyun Jin
- Department of Life Sciences, Korea University, Seoul 02841, Korea; (Z.N.); (K.G.); (H.S.); (S.J.); (G.Y.)
| | - Geummin Youn
- Department of Life Sciences, Korea University, Seoul 02841, Korea; (Z.N.); (K.G.); (H.S.); (S.J.); (G.Y.)
| | - Ji Hoon Ahn
- Department of Life Sciences, Korea University, Seoul 02841, Korea; (Z.N.); (K.G.); (H.S.); (S.J.); (G.Y.)
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27
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Singh AK, Zhang J, Hebenstreit D, Brogna S. Evidence of slightly increased Pol II pausing in UPF1-depleted Drosophila melanogaster cells. MICROPUBLICATION BIOLOGY 2020; 2020:10.17912/micropub.biology.000319. [PMID: 33274326 PMCID: PMC7704256 DOI: 10.17912/micropub.biology.000319] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Grants] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/12/2020] [Revised: 10/10/2020] [Accepted: 10/16/2020] [Indexed: 11/28/2022]
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28
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Ottesen EW, Singh RN. Characteristics of circular RNAs generated by human Survival Motor Neuron genes. Cell Signal 2020; 73:109696. [PMID: 32553550 PMCID: PMC7387165 DOI: 10.1016/j.cellsig.2020.109696] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2020] [Accepted: 06/12/2020] [Indexed: 02/06/2023]
Abstract
Circular RNAs (circRNAs) belong to a diverse class of stable RNAs expressed in all cell types. Their proposed functions include sponging of microRNAs (miRNAs), sequestration and trafficking of proteins, assembly of multimeric complexes, production of peptides, and regulation of transcription. Backsplicing due to RNA structures formed by an exceptionally high number of Alu repeats lead to the production of a vast repertoire of circRNAs by human Survival Motor Neuron genes, SMN1 and SMN2, that code for SMN, an essential multifunctional protein. Low levels of SMN due to deletion or mutation of SMN1 result in spinal muscular atrophy (SMA), a major genetic disease of infants and children. Mild SMA is also recorded in adult population, expanding the spectrum of the disease. Here we review SMN circRNAs with respect to their biogenesis, sequence features, and potential functions. We also discuss how SMN circRNAs could be exploited for diagnostic and therapeutic purposes.
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Affiliation(s)
- Eric W Ottesen
- Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, United States of America
| | - Ravindra N Singh
- Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, United States of America.
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29
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Domingo D, Nawaz U, Corbett M, Espinoza JL, Tatton-Brown K, Coman D, Wilkinson MF, Gecz J, Jolly LA. A synonymous UPF3B variant causing a speech disorder implicates NMD as a regulator of neurodevelopmental disorder gene networks. Hum Mol Genet 2020; 29:2568-2578. [PMID: 32667670 PMCID: PMC10893962 DOI: 10.1093/hmg/ddaa151] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 06/22/2020] [Accepted: 07/11/2020] [Indexed: 11/12/2022] Open
Abstract
Loss-of-function mutations of the X-chromosome gene UPF3B cause male neurodevelopmental disorders (NDDs) via largely unknown mechanisms. We investigated initially by interrogating a novel synonymous UPF3B variant in a male with absent speech. In silico and functional studies using cell lines derived from this individual show altered UPF3B RNA splicing. The resulting mRNA species encodes a frame-shifted protein with a premature termination codon (PTC) predicted to elicit degradation via nonsense-mediated mRNA decay (NMD). UPF3B mRNA was reduced in the cell line, and no UPF3B protein was produced, confirming a loss-of-function allele. UPF3B is itself involved in the NMD mechanism which degrades both PTC-bearing mutant transcripts and also many physiological transcripts. RNAseq analysis showed that ~1.6% of mRNAs exhibited altered expression. These mRNA changes overlapped and correlated with those we identified in additional cell lines obtained from individuals harbouring other UPF3B mutations, permitting us to interrogate pathogenic mechanisms of UPF3B-associated NDDs. We identified 102 genes consistently deregulated across all UPF3B mutant cell lines. Of the 51 upregulated genes, 75% contained an NMD-targeting feature, thus identifying high-confidence direct NMD targets. Intriguingly, 22 of the dysregulated genes encoded known NDD genes, suggesting UPF3B-dependent NMD regulates gene networks critical for cognition and behaviour. Indeed, we show that 78.5% of all NDD genes encode a transcript predicted to be targeted by NMD. These data describe the first synonymous UPF3B mutation in a patient with prominent speech and language disabilities and identify plausible mechanisms of pathology downstream of UPF3B mutations involving the deregulation of NDD-gene networks.
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Affiliation(s)
- Deepti Domingo
- University of Adelaide and Robinson Research Institute, Adelaide, SA 5005, Australia
| | - Urwah Nawaz
- University of Adelaide and Robinson Research Institute, Adelaide, SA 5005, Australia
| | - Mark Corbett
- University of Adelaide and Robinson Research Institute, Adelaide, SA 5005, Australia
| | | | - Katrina Tatton-Brown
- St George’s University of London, London SW17, UK
- Southwest Thames Regional Genetics Centre, St George’s Healthcare NHS Trust, London SW17, UK
| | - David Coman
- School of Medicine, University of Queensland, Brisbane, QLD 4072, Australia
| | - Miles F Wilkinson
- Department of Reproductive Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
- Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jozef Gecz
- University of Adelaide and Robinson Research Institute, Adelaide, SA 5005, Australia
- South Australian Health and Medical Research Institute, Adelaide, SA 5000, Australia
| | - Lachlan A Jolly
- University of Adelaide and Robinson Research Institute, Adelaide, SA 5005, Australia
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30
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Echols J, Siddiqui A, Dai Y, Havasi V, Sun R, Kaczmarczyk A, Keeling KM. A regulated NMD mouse model supports NMD inhibition as a viable therapeutic option to treat genetic diseases. Dis Model Mech 2020; 13:dmm044891. [PMID: 32737261 PMCID: PMC7473645 DOI: 10.1242/dmm.044891] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Accepted: 07/17/2020] [Indexed: 12/22/2022] Open
Abstract
Nonsense-mediated mRNA decay (NMD) targets mRNAs that contain a premature termination codon (PTC) for degradation, preventing their translation. By altering the expression of PTC-containing mRNAs, NMD modulates the inheritance pattern and severity of genetic diseases. NMD also limits the efficiency of suppressing translation termination at PTCs, an emerging therapeutic approach to treat genetic diseases caused by in-frame PTCs (nonsense mutations). Inhibiting NMD may help rescue partial levels of protein expression. However, it is unclear whether long-term, global NMD attenuation is safe. We hypothesize that a degree of NMD inhibition can be safely tolerated after completion of prenatal development. To test this hypothesis, we generated a novel transgenic mouse that expresses an inducible, dominant-negative form of human UPF1 (dnUPF1) to inhibit NMD in mouse tissues by different degrees, allowing us to examine the effects of global NMD inhibition in vivo A thorough characterization of these mice indicated that expressing dnUPF1 at levels that promote relatively moderate to strong NMD inhibition in most tissues for a 1-month period produced modest immunological and bone alterations. In contrast, 1 month of dnUPF1 expression to promote more modest NMD inhibition in most tissues did not produce any discernable defects, indicating that moderate global NMD attenuation is generally well tolerated in non-neurological somatic tissues. Importantly, a modest level of NMD inhibition that produced no overt abnormalities was able to significantly enhance in vivo PTC suppression. These results suggest that safe levels of NMD attenuation are likely achievable, and this can help rescue protein deficiencies resulting from PTCs.
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Affiliation(s)
- Josh Echols
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Amna Siddiqui
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Yanying Dai
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Viktoria Havasi
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Richard Sun
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Aneta Kaczmarczyk
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Kim M Keeling
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
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31
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Singh NN, Ottesen EW, Singh RN. A survey of transcripts generated by spinal muscular atrophy genes. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2020; 1863:194562. [PMID: 32387331 PMCID: PMC7302838 DOI: 10.1016/j.bbagrm.2020.194562] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Revised: 04/01/2020] [Accepted: 04/13/2020] [Indexed: 02/07/2023]
Abstract
Human Survival Motor Neuron (SMN) genes code for SMN, an essential multifunctional protein. Complete loss of SMN is embryonic lethal, while low levels of SMN lead to spinal muscular atrophy (SMA), a major genetic disease of children and infants. Reduced levels of SMN are associated with the abnormal development of heart, lung, muscle, gastro-intestinal system and testis. The SMN loci have been shown to generate a vast repertoire of transcripts, including linear, back- and trans-spliced RNAs as well as antisense long noncoding RNAs. However, functions of the majority of these transcripts remain unknown. Here we review the nature of RNAs generated from the SMN loci and discuss their potential functions in cellular metabolism.
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Affiliation(s)
- Natalia N Singh
- Department of Biomedical Science, Iowa State University, Ames, IA, 50011, United States of America
| | - Eric W Ottesen
- Department of Biomedical Science, Iowa State University, Ames, IA, 50011, United States of America
| | - Ravindra N Singh
- Department of Biomedical Science, Iowa State University, Ames, IA, 50011, United States of America.
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32
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Michalak M, Katzenmaier EM, Roeckel N, Woerner SM, Fuchs V, Warnken U, Yuan YP, Bork P, Neu-Yilik G, Kulozik A, von Knebel Doeberitz M, Kloor M, Kopitz J, Gebert J. (Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A. Int J Mol Sci 2020; 21:ijms21155234. [PMID: 32718059 PMCID: PMC7432364 DOI: 10.3390/ijms21155234] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2020] [Revised: 07/13/2020] [Accepted: 07/20/2020] [Indexed: 12/13/2022] Open
Abstract
DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.
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Affiliation(s)
- Malwina Michalak
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Department of Pediatric Oncology, Hematology and Immunology, Children’s Hospital, University of Heidelberg, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany
| | - Eva-Maria Katzenmaier
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
| | - Nina Roeckel
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
| | - Stefan M. Woerner
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Department of Internal Medicine I, Endocrinology and Metabolism, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
| | - Vera Fuchs
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
| | - Uwe Warnken
- Clinical Cooperation Unit Neurooncology, DKFZ (German Cancer Research Center), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany;
| | - Yan P. Yuan
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany;
| | - Peer Bork
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany;
- Max-Delbrück-Centre for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany
| | - Gabriele Neu-Yilik
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Department of Pediatric Oncology, Hematology and Immunology, Children’s Hospital, University of Heidelberg, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany
| | - Andreas Kulozik
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Department of Pediatric Oncology, Hematology and Immunology, Children’s Hospital, University of Heidelberg, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany
| | - Magnus von Knebel Doeberitz
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Clinical Cooperation Unit Applied Tumor Biology, DKFZ (German Cancer Research Center) Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - Matthias Kloor
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Clinical Cooperation Unit Applied Tumor Biology, DKFZ (German Cancer Research Center) Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - Jürgen Kopitz
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Clinical Cooperation Unit Applied Tumor Biology, DKFZ (German Cancer Research Center) Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - Johannes Gebert
- Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; (M.M.); (E.-M.K.); (N.R.); (V.F.); (M.v.K.D.); (M.K.); (J.K.)
- Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; (S.M.W.); (P.B.); (G.N.-Y.); (A.K.)
- Clinical Cooperation Unit Applied Tumor Biology, DKFZ (German Cancer Research Center) Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
- Correspondence: ; Tel.: +49-6221-564223
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33
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Lee HC, Kang D, Han N, Lee Y, Hwang HJ, Lee SB, You JS, Min BS, Park HJ, Ko YG, Gorospe M, Lee JS. A novel long noncoding RNA Linc-ASEN represses cellular senescence through multileveled reduction of p21 expression. Cell Death Differ 2020; 27:1844-1861. [PMID: 31819156 PMCID: PMC7244501 DOI: 10.1038/s41418-019-0467-6] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2019] [Revised: 11/20/2019] [Accepted: 11/21/2019] [Indexed: 01/10/2023] Open
Abstract
Long noncoding RNAs (lncRNAs) regulating diverse cellular processes implicate in many diseases. However, the function of lncRNAs in cellular senescence remains largely unknown. Here we identify a novel long intergenic noncoding RNA Linc-ASEN expresses in prematurely senescent cells. We find that Linc-ASEN associates with UPF1 by RNA pulldown mass spectrometry analysis, and represses cellular senescence by reducing p21 production transcriptionally and posttranscriptionally. Mechanistically, the Linc-ASEN-UPF1 complex suppressed p21 transcription by recruiting Polycomb Repressive Complex 1 (PRC1) and PRC2 to the p21 locus, and thereby preventing binding of the transcriptional activator p53 on the p21 promoter through histone modification. In addition, the Linc-ASEN-UPF1 complex repressed p21 expression posttranscriptionally by enhancing p21 mRNA decay in association with DCP1A. Accordingly, Linc-ASEN levels were found to correlate inversely with p21 mRNA levels in tumors from patient-derived mouse xenograft, in various human cancer tissues, and in aged mice tissues. Our results reveal that Linc-ASEN prevents cellular senescence by reducing the transcription and stability of p21 mRNA in concert with UPF1, and suggest that Linc-ASEN might be a potential therapeutic target in processes influenced by senescence, including cancer.
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Affiliation(s)
- Hyung Chul Lee
- Department of Molecular Medicine, and Medical Research Center, Inha University College of Medicine, Incheon, Korea
| | - Donghee Kang
- Department of Molecular Medicine, and Medical Research Center, Inha University College of Medicine, Incheon, Korea
| | - Namshik Han
- Milner Therapeutics Institute, University of Cambridge, Cambridge, UK
| | - Yerim Lee
- Department of Molecular Medicine, and Medical Research Center, Inha University College of Medicine, Incheon, Korea
| | - Hyun Jung Hwang
- Department of Molecular Medicine, and Medical Research Center, Inha University College of Medicine, Incheon, Korea
| | - Sat-Byol Lee
- Department of Surgery, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
| | - Jueng Soo You
- Department of Biochemistry, School of Medicine, Konkuk University, Seoul, Korea
| | - Byung Soh Min
- Department of Surgery, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
| | - Heon Joo Park
- Department of Microbiology, and Medical Research Center, Inha University College of Medicine, Incheon, Korea
| | - Young-Gyu Ko
- Division of Life Sciences, Korea University, Seoul, Korea
| | - Myriam Gorospe
- Laboratory of Genetics and Genomics, National Institute on Aging-Intramural Research Program, NIH, Baltimore, MD, USA
| | - Jae-Seon Lee
- Department of Molecular Medicine, and Medical Research Center, Inha University College of Medicine, Incheon, Korea.
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34
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Sharma J, Keeling KM, Rowe SM. Pharmacological approaches for targeting cystic fibrosis nonsense mutations. Eur J Med Chem 2020; 200:112436. [PMID: 32512483 DOI: 10.1016/j.ejmech.2020.112436] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2020] [Revised: 05/04/2020] [Accepted: 05/06/2020] [Indexed: 12/11/2022]
Abstract
Cystic fibrosis (CF) is a monogenic autosomal recessive disorder. The clinical manifestations of the disease are caused by ∼2,000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. It is unlikely that any one approach will be efficient in correcting all defects. The recent approvals of ivacaftor, lumacaftor/ivacaftor and elexacaftor/tezacaftor/ivacaftor represent the genesis of a new era of precision combination medicine for the CF patient population. In this review, we discuss targeted translational readthrough approaches as mono and combination therapies for CFTR nonsense mutations. We examine the current status of efficacy of translational readthrough/nonsense suppression therapies and their limitations, including non-native amino acid incorporation at PTCs and nonsense-mediated mRNA decay (NMD), along with approaches to tackle these limitations. We further elaborate on combining various therapies such as readthrough agents, NMD inhibitors, and corrector/potentiators to improve the efficacy and safety of suppression therapy. These mutation specific strategies that are directed towards the basic CF defects should positively impact CF patients bearing nonsense mutations.
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Affiliation(s)
- Jyoti Sharma
- Department of Medicine, University of Alabama at Birmingham (UAB), USA; Department of Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham (UAB), USA
| | - Kim M Keeling
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham (UAB), USA; Department of Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham (UAB), USA
| | - Steven M Rowe
- Department of Medicine, University of Alabama at Birmingham (UAB), USA; Department of Pediatrics, University of Alabama at Birmingham (UAB), USA; Department of Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham (UAB), USA.
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35
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Singh G, Fritz SE, Seufzer B, Boris-Lawrie K. The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR. J Biol Chem 2020; 295:7763-7773. [PMID: 32312751 DOI: 10.1074/jbc.ra119.012005] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Revised: 04/14/2020] [Indexed: 12/16/2022] Open
Abstract
One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of JUND (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of JUND RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT-quantitative PCR results revealed that JUND mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the JUND-containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of JUND-containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether JUND-NCBP1/NCBP3 to polysomes. We also found that JUND translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.
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Affiliation(s)
- Gatikrushna Singh
- Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, Minnesota 55108
| | - Sarah E Fritz
- Integrated Biomedical Science Graduate Program, Ohio State University, Columbus, Ohio 43210
| | - Bradley Seufzer
- Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, Minnesota 55108
| | - Kathleen Boris-Lawrie
- Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, Minnesota 55108 .,Integrated Biomedical Science Graduate Program, Ohio State University, Columbus, Ohio 43210
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36
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Keenan MM, Huang L, Jordan NJ, Wong E, Cheng Y, Valley HC, Mahiou J, Liang F, Bihler H, Mense M, Guo S, Monia BP. Nonsense-mediated RNA Decay Pathway Inhibition Restores Expression and Function of W1282X CFTR. Am J Respir Cell Mol Biol 2020; 61:290-300. [PMID: 30836009 DOI: 10.1165/rcmb.2018-0316oc] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The recessive genetic disease cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR (CF transmembrane conductance regulator) gene. Approximately 10% of patients with CF have at least one allele with a nonsense mutation in CFTR. Nonsense mutations generate premature termination codons that can subject mRNA transcripts to rapid degradation through the nonsense-mediated mRNA decay (NMD) pathway. Currently, there are no approved therapies that specifically target nonsense mutations in CFTR. Here, we identified antisense oligonucleotides (ASOs) that target the NMD factor SMG1 to inhibit the NMD pathway, and determined their effects on the W1282X CFTR mutation. First, we developed and validated two in vitro models of the W1282X CFTR mutation. Next, we treated these cells with antisense oligonucleotides to inhibit NMD and measured the effects of these treatments on W1282X expression and function. SMG1-ASO-mediated NMD inhibition upregulated the RNA, protein, and surface-localized protein expression of the truncated W1282X gene product. Additionally, these ASOs increased the CFTR chloride channel function in cells homozygous for the W1282X mutation. Our approach suggests a new therapeutic strategy for patients harboring nonsense mutations and may be beneficial as a single agent in patients with CF and the W1282X mutation.
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Affiliation(s)
| | - Lulu Huang
- Ionis Pharmaceuticals, Carlsbad, California; and
| | - Nikole J Jordan
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Eric Wong
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Yi Cheng
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Hillary C Valley
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Jerome Mahiou
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Feng Liang
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Hermann Bihler
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Martin Mense
- Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, Massachusetts
| | - Shuling Guo
- Ionis Pharmaceuticals, Carlsbad, California; and
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37
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Mino T, Iwai N, Endo M, Inoue K, Akaki K, Hia F, Uehata T, Emura T, Hidaka K, Suzuki Y, Standley DM, Okada-Hatakeyama M, Ohno S, Sugiyama H, Yamashita A, Takeuchi O. Translation-dependent unwinding of stem-loops by UPF1 licenses Regnase-1 to degrade inflammatory mRNAs. Nucleic Acids Res 2019; 47:8838-8859. [PMID: 31329944 PMCID: PMC7145602 DOI: 10.1093/nar/gkz628] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 07/05/2019] [Accepted: 07/12/2019] [Indexed: 01/14/2023] Open
Abstract
Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem–loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem–loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem–loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1–Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.
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Affiliation(s)
- Takashi Mino
- Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Noriki Iwai
- Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Masayuki Endo
- Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.,Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Kentaro Inoue
- Department of Computer Science and Systems Engineering, Faculty of Engineering, University of Miyazaki, Miyazaki 889-2192, Japan
| | - Kotaro Akaki
- Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Fabian Hia
- Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Takuya Uehata
- Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Tomoko Emura
- Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
| | - Kumi Hidaka
- Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
| | - Yutaka Suzuki
- Laboratory of Functional Genomics, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan
| | - Daron M Standley
- Department of Genome Informatics, Genome Information Research Center, Research Institute for Microbial Diseases (RIMD), Osaka University, Osaka 565-0871, Japan
| | - Mariko Okada-Hatakeyama
- Laboratory for Integrated Cellular Systems, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan.,Laboratory of Cell Systems, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan
| | - Shigeo Ohno
- Department of Molecular Biology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan
| | - Hiroshi Sugiyama
- Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.,Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Akio Yamashita
- Department of Molecular Biology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan
| | - Osamu Takeuchi
- Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
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Belloni E, Di Matteo A, Pradella D, Vacca M, Wyatt CDR, Alfieri R, Maffia A, Sabbioneda S, Ghigna C. Gene Expression Profiles Controlled by the Alternative Splicing Factor Nova2 in Endothelial Cells. Cells 2019; 8:cells8121498. [PMID: 31771184 PMCID: PMC6953062 DOI: 10.3390/cells8121498] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 11/11/2019] [Accepted: 11/20/2019] [Indexed: 02/07/2023] Open
Abstract
Alternative splicing (AS) plays an important role in expanding the complexity of the human genome through the production of specialized proteins regulating organ development and physiological functions, as well as contributing to several pathological conditions. How AS programs impact on the signaling pathways controlling endothelial cell (EC) functions and vascular development is largely unknown. Here we identified, through RNA-seq, changes in mRNA steady-state levels in ECs caused by the neuro-oncological ventral antigen 2 (Nova2), a key AS regulator of the vascular morphogenesis. Bioinformatics analyses identified significant enrichment for genes regulated by peroxisome proliferator-activated receptor-gamma (Ppar-γ) and E2F1 transcription factors. We also showed that Nova2 in ECs controlled the AS profiles of Ppar-γ and E2F dimerization partner 2 (Tfdp2), thus generating different protein isoforms with distinct function (Ppar-γ) or subcellular localization (Tfdp2). Collectively, our results supported a mechanism whereby Nova2 integrated splicing decisions in order to regulate Ppar-γ and E2F1 activities. Our data added a layer to the sequential series of events controlled by Nova2 in ECs to orchestrate vascular biology.
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Affiliation(s)
- Elisa Belloni
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
| | - Anna Di Matteo
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
| | - Davide Pradella
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
| | - Margherita Vacca
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
| | - Christopher D. R. Wyatt
- Centre for Biodiversity and Environment Research, University College London, Gower Street, London WC1E 6BT, UK
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr Aiguader 88, 08003 Barcelona, Spain
- Universitat Pompeu Fabra, Plaça de la Mercè, 10-12, 08002 Barcelona, Spain
| | - Roberta Alfieri
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
| | - Antonio Maffia
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
| | - Simone Sabbioneda
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
| | - Claudia Ghigna
- Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy; (E.B.); (A.D.M.); (D.P.); (M.V.); (R.A.); (A.M.); (S.S.)
- Correspondence:
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Zhao B, Pritchard JR. Evolution of the nonsense-mediated decay pathway is associated with decreased cytolytic immune infiltration. PLoS Comput Biol 2019; 15:e1007467. [PMID: 31658270 PMCID: PMC6837539 DOI: 10.1371/journal.pcbi.1007467] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2019] [Revised: 11/07/2019] [Accepted: 10/08/2019] [Indexed: 01/05/2023] Open
Abstract
The somatic co-evolution of tumors and the cellular immune responses that combat them drives the diversity of immune-tumor interactions. This includes tumor mutations that generate neo-antigenic epitopes that elicit cytotoxic T-cell activity and subsequent pressure to select for genetic loss of antigen presentation. Most studies have focused on how tumor missense mutations can drive tumor immunity, but frameshift mutations have the potential to create far greater antigenic diversity. However, expression of this antigenic diversity is potentially regulated by Nonsense Mediated Decay (NMD) and NMD has been shown to be of variable efficiency in cancers. Here we studied how mutational changes influence global NMD and cytolytic immune responses. Using TCGA datasets, we derived novel patient-level metrics of 'NMD burden' and interrogated how different mutation and most importantly NMD burdens influence cytolytic activity using machine learning models and survival outcomes. We find that NMD is a significant and independent predictor of immune cytolytic activity. Different indications exhibited varying dependence on NMD and mutation burden features. We also observed significant co-alteration of genes in the NMD pathway, with a global increase in NMD efficiency in patients with NMD co-alterations. Finally, NMD burden also stratified patient survival in multivariate regression models in subset of cancer types. Our work suggests that beyond selecting for mutations that elicit NMD in tumor suppressors, tumor evolution may react to the selective pressure generated by inflammation to globally enhance NMD through coordinated amplification and/or mutation.
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Affiliation(s)
- Boyang Zhao
- Department of Biomedical Engineering, College of Engineering, The Pennsylvania State University, University Park, Pennsylvania, United States of America
- Quantalarity Research Group LLC, Houston, Texas, United States of America
| | - Justin R. Pritchard
- Department of Biomedical Engineering, College of Engineering, The Pennsylvania State University, University Park, Pennsylvania, United States of America
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Tam BY, Chiu K, Chung H, Bossard C, Nguyen JD, Creger E, Eastman BW, Mak CC, Ibanez M, Ghias A, Cahiwat J, Do L, Cho S, Nguyen J, Deshmukh V, Stewart J, Chen CW, Barroga C, Dellamary L, Kc SK, Phalen TJ, Hood J, Cha S, Yazici Y. The CLK inhibitor SM08502 induces anti-tumor activity and reduces Wnt pathway gene expression in gastrointestinal cancer models. Cancer Lett 2019; 473:186-197. [PMID: 31560935 DOI: 10.1016/j.canlet.2019.09.009] [Citation(s) in RCA: 97] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2019] [Revised: 09/12/2019] [Accepted: 09/17/2019] [Indexed: 02/07/2023]
Abstract
The Wnt/β-catenin signaling pathway is aberrantly activated in colorectal (CRC) and many other cancers, and novel strategies for effectively targeting it may be needed due to its complexity. In this report, SM08502, a novel small molecule in clinical development for the treatment of solid tumors, was shown to reduce Wnt pathway signaling and gene expression through potent inhibition of CDC-like kinase (CLK) activity. SM08502 inhibited serine and arginine rich splicing factor (SRSF) phosphorylation and disrupted spliceosome activity, which was associated with inhibition of Wnt pathway-related gene and protein expression. Additionally, SM08502 induced the generation of splicing variants of Wnt pathway genes, suggesting that its mechanism for inhibition of gene expression includes effects on alternative splicing. Orally administered SM08502 significantly inhibited growth of gastrointestinal tumors and decreased SRSF phosphorylation and Wnt pathway gene expression in xenograft mouse models. These data implicate CLKs in the regulation of Wnt signaling and represent a novel strategy for inhibiting Wnt pathway gene expression in cancers. SM08502 is a first-in-class CLK inhibitor being investigated in a Phase 1 clinical trial for subjects with advanced solid tumors (NCT03355066).
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | - Long Do
- Samumed, LLC, San Diego, CA, USA
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Deshmukh V, O'Green AL, Bossard C, Seo T, Lamangan L, Ibanez M, Ghias A, Lai C, Do L, Cho S, Cahiwat J, Chiu K, Pedraza M, Anderson S, Harris R, Dellamary L, Kc S, Barroga C, Melchior B, Tam B, Kennedy S, Tambiah J, Hood J, Yazici Y. Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment. Osteoarthritis Cartilage 2019; 27:1347-1360. [PMID: 31132406 DOI: 10.1016/j.joca.2019.05.006] [Citation(s) in RCA: 126] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 04/23/2019] [Accepted: 05/14/2019] [Indexed: 02/02/2023]
Abstract
OBJECTIVES Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteoblast differentiation, increased catabolic enzymes, and inflammation. The small-molecule Wnt pathway inhibitor, lorecivivint (SM04690), which previously demonstrated chondrogenesis and cartilage protection in an animal OA model, was evaluated to elucidate its mechanism of action. DESIGN Biochemical assays measured kinase activity. Western blots measured protein phosphorylation in human mesenchymal stem cells (hMSCs), chondrocytes, and synovial fibroblasts. siRNA knockdown effects in hMSCs and BEAS-2B cells on Wnt pathway, chondrogenic genes, and LPS-induced inflammatory cytokines was measured by qPCR. In vivo anti-inflammation, pain, and function were evaluated following single intra-articular (IA) lorecivivint or vehicle injection in the monosodium iodoacetate (MIA)-induced rat OA model. RESULTS Lorecivivint inhibited intranuclear kinases CDC-like kinase 2 (CLK2) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Lorecivivint inhibited CLK2-mediated phosphorylation of serine/arginine-rich (SR) splicing factors and DYRK1A-mediated phosphorylation of SIRT1 and FOXO1. siRNA knockdowns identified a role for CLK2 and DYRK1A in Wnt pathway modulation without affecting β-catenin with CLK2 inhibition inducing early chondrogenesis and DYRK1A inhibition enhancing mature chondrocyte function. NF-κB and STAT3 inhibition by lorecivivint reduced inflammation. DYRK1A knockdown was sufficient for anti-inflammatory effects, while combined DYRK1A/CLK2 knockdown enhanced this effect. In the MIA model, lorecivivint inhibited production of inflammatory cytokines and cartilage degradative enzymes, resulting in increased joint cartilage, decreased pain, and improved weight-bearing function. CONCLUSIONS Lorecivivint inhibition of CLK2 and DYRK1A suggested a novel mechanism for Wnt pathway inhibition, enhancing chondrogenesis, chondrocyte function, and anti-inflammation. Lorecivivint shows potential to modify structure and improve symptoms of knee OA.
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Affiliation(s)
| | | | | | - T Seo
- Samumed, LLC, San Diego, CA, USA.
| | | | - M Ibanez
- Samumed, LLC, San Diego, CA, USA.
| | - A Ghias
- Samumed, LLC, San Diego, CA, USA.
| | - C Lai
- Samumed, LLC, San Diego, CA, USA.
| | - L Do
- Samumed, LLC, San Diego, CA, USA.
| | - S Cho
- Samumed, LLC, San Diego, CA, USA.
| | | | - K Chiu
- Samumed, LLC, San Diego, CA, USA.
| | | | | | - R Harris
- Samumed, LLC, San Diego, CA, USA.
| | | | - S Kc
- Samumed, LLC, San Diego, CA, USA.
| | | | | | - B Tam
- Formerly Samumed, LLC, USA.
| | | | | | - J Hood
- Formerly Samumed, LLC, USA.
| | - Y Yazici
- Samumed, LLC, San Diego, CA, USA.
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42
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CSL controls telomere maintenance and genome stability in human dermal fibroblasts. Nat Commun 2019; 10:3884. [PMID: 31467287 PMCID: PMC6715699 DOI: 10.1038/s41467-019-11785-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2019] [Accepted: 07/31/2019] [Indexed: 12/31/2022] Open
Abstract
Genomic instability is a hallmark of cancer. Whether it also occurs in Cancer Associated Fibroblasts (CAFs) remains to be carefully investigated. Loss of CSL/RBP-Jκ, the effector of canonical NOTCH signaling with intrinsic transcription repressive function, causes conversion of dermal fibroblasts into CAFs. Here, we find that CSL down-modulation triggers DNA damage, telomere loss and chromosome end fusions that also occur in skin Squamous Cell Carcinoma (SCC)-associated CAFs, in which CSL is decreased. Separately from its role in transcription, we show that CSL is part of a multiprotein telomere protective complex, binding directly and with high affinity to telomeric DNA as well as to UPF1 and Ku70/Ku80 proteins and being required for their telomere association. Taken together, the findings point to a central role of CSL in telomere homeostasis with important implications for genomic instability of cancer stromal cells and beyond. Conversion of dermal fibroblasts into Cancer Associated Fibroblasts (CAFs) can play an important role in keratinocyte tumour development. Here the authors reveal that CSL plays a role in maintenance of telomeres and genomic integrity in both dermal fibroblasts and CAFs.
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43
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Hong D, Park T, Jeong S. Nuclear UPF1 Is Associated with Chromatin for Transcription-Coupled RNA Surveillance. Mol Cells 2019; 42:523-529. [PMID: 31234619 PMCID: PMC6681869 DOI: 10.14348/molcells.2019.0116] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2019] [Revised: 06/19/2019] [Accepted: 06/19/2019] [Indexed: 01/26/2023] Open
Abstract
mRNA quality is controlled by multiple RNA surveillance machineries to reduce errors during gene expression processes in eukaryotic cells. Nonsense-mediated mRNA decay (NMD) is a well-characterized mechanism that degrades error-containing transcripts during translation. The ATP-dependent RNA helicase up-frameshift 1 (UPF1) is a key player in NMD that is mostly prevalent in the cytoplasm. However, recent studies on UPF1-RNA interaction suggest more comprehensive roles of UPF1 on diverse forms of target transcripts. Here we used subcellular fractionation and immunofluorescence to understand such complex functions of UPF1. We demonstrated that UPF1 can be localized to the nucleus and predominantly associated with the chromatin. Moreover, we showed that UPF1 associates more strongly with the chromatin when the transcription elongation and translation inhibitors were used. These findings suggest a novel role of UPF1 in transcription elongation-coupled RNA machinery in the chromatin, as well as in translation-coupled NMD in the cytoplasm. Thus, we propose that cytoplasmic UPF1-centric RNA surveillance mechanism could be extended further up to the chromatin-associated UPF1 and cotranscriptional RNA surveillance. Our findings could provide the mechanistic insights on extensive regulatory roles of UPF1 for many cellular RNAs.
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Affiliation(s)
- Dawon Hong
- Graduate Department of Bioconvergence Science and Technology, Dankook University, Yongin 16892,
Korea
| | - Taeyoung Park
- Graduate Department of Bioconvergence Science and Technology, Dankook University, Yongin 16892,
Korea
| | - Sunjoo Jeong
- Graduate Department of Bioconvergence Science and Technology, Dankook University, Yongin 16892,
Korea
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Hocq R, Paternina J, Alasseur Q, Genovesio A, Le Hir H. Monitored eCLIP: high accuracy mapping of RNA-protein interactions. Nucleic Acids Res 2019; 46:11553-11565. [PMID: 30252095 PMCID: PMC6265473 DOI: 10.1093/nar/gky858] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2018] [Accepted: 09/13/2018] [Indexed: 01/29/2023] Open
Abstract
CLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the ‘monitored enhanced CLIP’ (meCLIP) method, a barcoded biotinylated linker is ligated at the 5′ end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence correspond to reverse transcription read-throughs. Read through occurs in unpredictable proportions, representing up to one fourth of total reads. Filtering out those reads strongly improves reliability and precision in protein binding site assignment.
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Affiliation(s)
- Rémi Hocq
- Institut de biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS UMR8197, INSERM U1024, PSL Research University, 75005 Paris, France
| | - Janio Paternina
- Institut de biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS UMR8197, INSERM U1024, PSL Research University, 75005 Paris, France
| | - Quentin Alasseur
- Institut de biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS UMR8197, INSERM U1024, PSL Research University, 75005 Paris, France
| | - Auguste Genovesio
- Institut de biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS UMR8197, INSERM U1024, PSL Research University, 75005 Paris, France
| | - Hervé Le Hir
- Institut de biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS UMR8197, INSERM U1024, PSL Research University, 75005 Paris, France
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Ferreira PA. The coming-of-age of nucleocytoplasmic transport in motor neuron disease and neurodegeneration. Cell Mol Life Sci 2019; 76:2247-2273. [PMID: 30742233 PMCID: PMC6531325 DOI: 10.1007/s00018-019-03029-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 01/28/2019] [Indexed: 12/11/2022]
Abstract
The nuclear pore is the gatekeeper of nucleocytoplasmic transport and signaling through which a vast flux of information is continuously exchanged between the nuclear and cytoplasmic compartments to maintain cellular homeostasis. A unifying and organizing principle has recently emerged that cements the notion that several forms of amyotrophic lateral sclerosis (ALS), and growing number of other neurodegenerative diseases, co-opt the dysregulation of nucleocytoplasmic transport and that this impairment is a pathogenic driver of neurodegeneration. The understanding of shared pathomechanisms that underpin neurodegenerative diseases with impairments in nucleocytoplasmic transport and how these interface with current concepts of nucleocytoplasmic transport is bound to illuminate this fundamental biological process in a yet more physiological context. Here, I summarize unresolved questions and evidence and extend basic and critical concepts and challenges of nucleocytoplasmic transport and its role in the pathogenesis of neurodegenerative diseases, such as ALS. These principles will help to appreciate the roles of nucleocytoplasmic transport in the pathogenesis of ALS and other neurodegenerative diseases, and generate a framework for new ideas of the susceptibility of motoneurons, and possibly other neurons, to degeneration by dysregulation of nucleocytoplasmic transport.
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Affiliation(s)
- Paulo A Ferreira
- Duke University Medical Center, DUEC 3802, 2351 Erwin Road, Durham, NC, 27710, USA.
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46
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Aksit MA, Bowling AD, Evans TA, Joynt AT, Osorio D, Patel S, West N, Merlo C, Sosnay PR, Cutting GR, Sharma N. Decreased mRNA and protein stability of W1282X limits response to modulator therapy. J Cyst Fibros 2019; 18:606-613. [PMID: 30803905 DOI: 10.1016/j.jcf.2019.02.009] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Revised: 02/14/2019] [Accepted: 02/14/2019] [Indexed: 12/13/2022]
Abstract
BACKGROUND Cell-based studies have shown that W1282X generates a truncated protein that can be functionally augmented by modulators. However, modulator treatment of primary cells from individuals who carry two copies of W1282X generates no functional CFTR. To understand the lack of response to modulators, we investigated the effect of W1282X on CFTR RNA transcript levels. METHODS qRT-PCR and RNA-seq were performed on primary nasal epithelial (NE) cells of a previously studied individual who is homozygous for W1282X, her carrier parents and control individuals without nonsense variants in CFTR. RESULTS CFTR RNA bearing W1282X in NE cells shows a steady-state level of 4.2 ± 0.9% of wild-type (WT) CFTR RNA in the mother and 12.4 ± 1.3% in the father. NMDI14, an inhibitor of nonsense-mediated mRNA decay (NMD), restored W1282X mRNA to almost 50% of WT levels in the parental NE cells. RNA-seq of the NE cells homozygous for W1282X showed that CFTR transcript level was reduced to 1.7% of WT (p-value: 4.6e-3). Negligible truncated CFTR protein was generated by Flp-In 293 cells stably expressing the W1282X EMG even though CFTR transcript was well above levels observed in the parents and proband. Finally, we demonstrated that NMD inhibition improved the stability and response to correctors of W1282X-CFTR protein expressed in the Flp-In-293 cells. CONCLUSION These results show that W1282X can cause substantial degradation of CFTR mRNA that has to be addressed before efforts aimed at augmenting CFTR protein function can be effective.
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Affiliation(s)
- M A Aksit
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - A D Bowling
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - T A Evans
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - A T Joynt
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - D Osorio
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - S Patel
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, United States
| | - N West
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, United States
| | - C Merlo
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, United States
| | - P R Sosnay
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, United States
| | - G R Cutting
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - N Sharma
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States.
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Fike AJ, Elcheva I, Rahman ZSM. The Post-GWAS Era: How to Validate the Contribution of Gene Variants in Lupus. Curr Rheumatol Rep 2019; 21:3. [DOI: 10.1007/s11926-019-0801-5] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
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Whitehouse LLE, Smith CEL, Poulter JA, Brown CJ, Patel A, Lamb T, Brown LR, O’Sullivan EA, Mitchell RE, Berry IR, Charlton R, Inglehearn CF, Mighell AJ. Novel DLX3 variants in amelogenesis imperfecta with attenuated tricho-dento-osseous syndrome. Oral Dis 2019; 25:182-191. [PMID: 30095208 PMCID: PMC6334507 DOI: 10.1111/odi.12955] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2018] [Revised: 07/12/2018] [Accepted: 08/03/2018] [Indexed: 12/20/2022]
Abstract
OBJECTIVES Variants in DLX3 cause tricho-dento-osseous syndrome (TDO, MIM #190320), a systemic condition with hair, nail and bony changes, taurodontism and amelogenesis imperfecta (AI), inherited in an autosomal dominant fashion. Different variants found within this gene are associated with different phenotypic presentations. To date, six different DLX3 variants have been reported in TDO. The aim of this paper was to explore and discuss three recently uncovered new variants in DLX3. SUBJECTS AND METHODS Whole-exome sequencing identified a new DLX3 variant in one family, recruited as part of an ongoing study of genetic variants associated with AI. Targeted clinical exome sequencing of two further families revealed another new variant of DLX3 and complete heterozygous deletion of DLX3. For all three families, the phenotypes were shown to consist of AI and taurodontism, together with other attenuated features of TDO. RESULTS c.574delG p.(E192Rfs*66), c.476G>T (p.R159L) and a heterozygous deletion of the entire DLX3 coding region were identified in our families. CONCLUSION These previously unreported variants add to the growing literature surrounding AI, allowing for more accurate genetic testing and better understanding of the associated clinical consequences.
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Affiliation(s)
| | - Claire E. L. Smith
- Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical SciencesUniversity of LeedsLeedsUK
| | | | | | - Anesha Patel
- Birmingham Dental Hospital and School of DentistryBirminghamUK
| | - Teresa Lamb
- Oxford University Hospitals NHS Foundation TrustOxfordUK
| | | | | | | | - Ian R. Berry
- Leeds Genetics LaboratorySt James’s University HospitalLeedsUK
| | - Ruth Charlton
- Leeds Genetics LaboratorySt James’s University HospitalLeedsUK
| | - Chris F. Inglehearn
- Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical SciencesUniversity of LeedsLeedsUK
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Hernando CE, Garcia C, Mateos JL. Casting Away the Shadows: Elucidating the Role of Light-mediated Posttranscriptional Control in Plants. Photochem Photobiol 2018; 93:656-665. [PMID: 28500720 DOI: 10.1111/php.12762] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2016] [Accepted: 02/15/2017] [Indexed: 12/21/2022]
Abstract
Light signals trigger precise changes in gene expression networks that activate distinctive developmental programs in plants. The transcriptome is shaped at different stages, both by the regulation of gene expression and also by posttranscriptional mechanisms that alter the sequence or abundance of the transcripts generated. Posttranscriptional mechanisms have attracted much interest in recent years with the advent of high-throughput technologies and bioinformatics tools. One such posttranscriptional process, alternative splicing, increases proteome diversity without increasing gene number by changing the function of individual proteins, while another, miRNA-mediated gene silencing, fine-tunes the amount of mRNA produced. The manner in which plants make use of these two crucial posttranscriptional mechanisms to respond to light and adapt to their environment is the focus of active research. In this review, we summarize the current knowledge of light-mediated posttranscriptional control in Arabidopsis thaliana and focus on the biological impact of the various posttranscriptional processes. We also discuss a potential cross talk between the alternative splicing and miRNA pathways, highlighting the complexity of light responsiveness.
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Affiliation(s)
| | - Carolina Garcia
- Fundación Instituto Leloir, IIBBA-CONICET, Buenos Aires, Argentina
| | - Julieta L Mateos
- Fundación Instituto Leloir, IIBBA-CONICET, Buenos Aires, Argentina
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Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway. Proc Natl Acad Sci U S A 2018; 115:E10157-E10166. [PMID: 30297408 PMCID: PMC6205489 DOI: 10.1073/pnas.1811675115] [Citation(s) in RCA: 64] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Coronaviruses (CoVs) are important pathogens for humans and domestic animals. The development of effective countermeasures against CoVs requires an understanding of the host pathways that regulate viral gene expression and the viral subversion mechanisms. However, little is known about how the stability of viral mRNAs is controlled. We show that the nonsense-mediated decay (NMD) pathway, which primarily targets aberrant cellular mRNAs for degradation, also induced the degradation of CoV mRNAs that are of cytoplasmic origin. Our study further suggests the importance of CoV-induced inhibition of the NMD pathway, mediated by a viral protein, for efficient CoV replication. The present study highlights an interplay between the NMD pathway and CoVs that modulates viral replication by controlling the stability of viral mRNAs. Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Very little is known about the host pathways that regulate the stability of CoV mRNAs, which carry some unusual features. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation. Although CoV mRNAs are of cytoplasmic origin, the presence of several NMD-inducing features (including multiple ORFs with internal termination codons that create a long 3′ untranslated region) in CoV mRNAs led us to explore the interplay between the NMD pathway and CoVs. Our study using murine hepatitis virus as a model CoV showed that CoV mRNAs are recognized by the NMD pathway as a substrate, resulting in their degradation. Furthermore, CoV replication induced the inhibition of the NMD pathway, and N protein (a viral structural protein) had an NMD inhibitory function that protected viral mRNAs from rapid decay. Our data further suggest that the NMD pathway interferes with optimal viral replication by degrading viral mRNAs early in infection, before sufficient accumulation of N protein. Our study presents clear evidence for the biological importance of the NMD pathway in controlling the stability of mRNAs and the efficiency of replication of a cytoplasmic RNA virus.
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