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1
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Fernandez M, Mendez R. Cytoplasmic regulation of the poly(A) tail length as a potential therapeutic target. RNA (NEW YORK, N.Y.) 2025; 31:402-415. [PMID: 39805658 PMCID: PMC11874964 DOI: 10.1261/rna.080333.124] [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: 11/21/2024] [Accepted: 12/27/2024] [Indexed: 01/16/2025]
Abstract
Virtually all mRNAs acquire a poly(A) tail cotranscriptionally, but its length is dynamically regulated in the cytoplasm in a transcript-specific manner. The length of the poly(A) tail plays a crucial role in determining mRNA translation, stability, and localization. This dynamic regulation of poly(A) tail length is widely used to create posttranscriptional gene expression programs, allowing for precise temporal and spatial control. Dysregulation of poly(A) tail length has been linked to various diseases, including cancers, inflammatory and cardiovascular disorders, and neurological syndromes. Cytoplasmic poly(A) tail length is maintained by a dynamic equilibrium between cis-acting elements and cognate factors that promote deadenylation or polyadenylation, enabling rapid gene expression reprogramming in response to internal and external cellular cues. While cytoplasmic deadenylation and its pathophysiological implications have been extensively studied, cytoplasmic polyadenylation and its therapeutic potential remain less explored. This review discusses the distribution, regulation, and mechanisms of cytoplasmic polyadenylation element-binding proteins(CPEBs), highlighting their dual roles in either promoting or repressing gene expression depending on cellular context. We also explore their involvement in diseases such as tumor progression and metastasis, along with their potential as targets for novel therapeutic strategies.
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Affiliation(s)
| | - Raul Mendez
- Institute for Research in Biomedicine (IRB), The Barcelona Institute of Science and Technology, 08028 Barcelona, Spain
- Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain
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2
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Chilukuri A, Kim M, Mitra T, Gubatan JM, Urrete J, Saxon LD, Ablack A, Mikulski Z, Dobaczewska K, Shen Z, Keir M, Yi T, Kaur P, Oliveira P, Murillo-Saich J, Chang EY, Steiner CA, Jedlicka P, Guma M, Rivera-Nieves J. A Similar Mutation in the AAUU-Rich Elements of the Mouse TNF Gene Results in a Distinct Ileocolitic Phenotype: A New Strain of TNF-Overexpressing Mice. Inflamm Bowel Dis 2025:izae307. [PMID: 39756463 DOI: 10.1093/ibd/izae307] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Indexed: 01/07/2025]
Abstract
BACKGROUND Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays a critical role in the pathogenesis of immune-mediated diseases including inflammatory bowel disease (IBD). The stability of its mRNA transcript, determined in part by destabilizing sequences in its AAUU repeats (ARE) gene region, is an important regulator of its tissue and systemic levels. A deletion in the ARE region of the gene resulted in IBD and arthritis in mice and pigs, supporting a critical role for the cytokine in human IBD and several human arthritides. A mutation in the same area of the mouse genome by Genentech scientists (T.Y., M.K.) resulted in a similar but not identical phenotype. METHODS Here, we compare histopathological, cellular, and molecular features of the strains and propose reasons for their distinct phenotypes. First, while homozygous TNFΔARE mice develop severe arthritis and die after weaning, homozygous Genentech TNFΔARE (ΔG/ΔG) mice have normal lifespans, and males are often fertile. RESULTS We found that while the ileitic phenotype had peaked at 12 weeks of age in all mice, colitis progressed mostly after 20 weeks of age in heterozygous mice. Their variably penetrant arthritic phenotype progressed mostly after 20 weeks, also in heterozygous mice from both strains. There was expansion of central memory T and B cells in lymphoid organs of TNF-overproducing strains and their transcriptional profile shared well-known pathogenetic pathways with human IBD. Finally, we found differences in the mutated sequences within the ARE regions of the TNF gene and in their microbiota composition and genetic background. These differences likely explain their phenotypic differences. CONCLUSIONS In summary, we describe a different strain of TNF-overproducing mice with an overlapping, yet not identical phenotype, which may have differential applications than the original strain.
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Affiliation(s)
- Amruth Chilukuri
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
| | - Margaret Kim
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
- Gastroenterology Section, San Diego VA Medical Center, La Jolla Village Drive, San Diego, CA, USA
| | - Taniya Mitra
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
- Gastroenterology Section, San Diego VA Medical Center, La Jolla Village Drive, San Diego, CA, USA
| | - John M Gubatan
- Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA
| | - Josef Urrete
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
| | - Leo D Saxon
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
- Gastroenterology Section, San Diego VA Medical Center, La Jolla Village Drive, San Diego, CA, USA
| | - Amber Ablack
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
| | - Zbigniew Mikulski
- Microscopy and Histology Core, La Jolla Institute of Allergy and Immunology, La Jolla, CA, USA
| | - Katarzyna Dobaczewska
- Microscopy and Histology Core, La Jolla Institute of Allergy and Immunology, La Jolla, CA, USA
| | - Zining Shen
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
| | - Mary Keir
- Genentech Pharmaceuticals, South San Francisco, CA, USA
| | - Tangsheng Yi
- Genentech Pharmaceuticals, South San Francisco, CA, USA
| | - Prabhdeep Kaur
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
- Gastroenterology Section, San Diego VA Medical Center, La Jolla Village Drive, San Diego, CA, USA
| | - Patricia Oliveira
- Rheumatology Division, University of California San Diego, La Jolla, CA, USA
| | | | - Eric Y Chang
- Radiology Department, San Diego VA Medical Center, La Jolla Village Drive, San Diego, CA, USA
| | - Calen A Steiner
- Division of Gastroenterology, University of Colorado, Denver, CO, USA
| | - Paul Jedlicka
- Department of Pathology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA
| | - Mónica Guma
- Rheumatology Division, University of California San Diego, La Jolla, CA, USA
| | - Jesús Rivera-Nieves
- Division of Gastroenterology, University of California San Diego, La Jolla, CA, USA
- Gastroenterology Section, San Diego VA Medical Center, La Jolla Village Drive, San Diego, CA, USA
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Zhang L, Chen Z, Sun G, Li C, Wu P, Xu W, Zhu H, Zhang Z, Tang Y, Li Y, Li Y, Xu S, Li H, Chen M, Xiao F, Zhang Y, Zhang W. Dynamic landscape of m6A modifications and related post-transcriptional events in muscle-invasive bladder cancer. J Transl Med 2024; 22:912. [PMID: 39380003 PMCID: PMC11460118 DOI: 10.1186/s12967-024-05701-x] [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: 08/05/2024] [Accepted: 09/23/2024] [Indexed: 10/10/2024] Open
Abstract
BACKGROUND Muscle-invasive bladder carcinoma (MIBC) is a serious and more advanced stage of bladder carcinoma. N6-Methyladenosine (m6A) is a dynamic and reversible modifications that primarily affects RNA stability and alternative splicing. The dysregulation of m6A in MIBC can be potential target for clinical interventions, but there have been limited studies on m6A modifications in MIBC and their associations with post-transcriptional regulatory processes. METHODS Paired tumor and adjacent-normal tissues were obtained from three patients with MIBC following radical cystectomy. The additional paired tissues for validation were obtained from patients underwent transurethral resection. Utilizing Nanopore direct-RNA sequencing, we characterized the m6A RNA methylation landscape in MIBC, with a focus on identifying post-transcriptional events potentially affected by changes in m6A sites. This included an examination of differential transcript usage, polyadenylation signal sites, and variations in poly(A) tail length, providing insights into the broader impact of m6A alterations on RNA processing in MIBC. RESULTS The prognostic-related m6A genes and m6A-risk model constructed by machine learning enables the stratification of high and low-risk patients with precision. A novel m6A modification site in the 3' untranslated region (3'UTR) of IGLL5 gene were identified, characterized by a lower m6A methylation ratio, elongated poly(A) tails, and a notable bias in transcript usage. Furthermore, we discovered two particular transcripts, VWA1-203 and CEBPB-201. VWA1-203 displayed diminished m6A methylation levels, a truncated 3'UTR, and an elongated poly(A) tail, whereas CEBPB-201 showed opposite trends, highlighting the complex interplay between m6A modifications and RNA processing. Source code was provided on GitHub ( https://github.com/lelelililele/Nanopore-m6A-analysis ). CONCLUSIONS The state-of-the-art Nanopore direct-RNA sequencing and machine learning techniques enables comprehensive identification of m6A modification and provided insights into the potential post-transcriptional regulation mechanisms on the development and progression in MIBC.
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Affiliation(s)
- Lili Zhang
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Ziwei Chen
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
- Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Gaoyuan Sun
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Chang Li
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Pengjie Wu
- Department of Urology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Wenrui Xu
- Department of Radiology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, P.R. China
| | - Hui Zhu
- Department of Nuclear Medicine, Beijing Hospital, National Center of Gerontology, Beijing, China
| | - Zaifeng Zhang
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
- Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Yongbin Tang
- Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
- Department of Urology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Yayu Li
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
- Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
- University of Chinese Academy of Sciences Medical School, Beijing, China
| | - Yifei Li
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Siyuan Xu
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Hexin Li
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China
| | - Meng Chen
- National Clinical Research Center for Cancer/Cancer Hospital, National Cancer Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Fei Xiao
- Clinical Biobank, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China.
- Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
| | - Yaqun Zhang
- Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, Tianjin, China.
| | - Wei Zhang
- Department of Pathology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China.
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Stedile M, Lara Montero A, García Solá ME, Goddio MV, Beckerman I, Bogni E, Ayre M, Naguila Z, Coso OA, Kordon EC. Tristetraprolin promotes survival of mammary progenitor cells by restraining TNFα levels. Front Cell Dev Biol 2024; 11:1265475. [PMID: 38274271 PMCID: PMC10808302 DOI: 10.3389/fcell.2023.1265475] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2023] [Accepted: 12/22/2023] [Indexed: 01/27/2024] Open
Abstract
Tristetraprolin (TTP) is an RNA binding protein that destabilizes mRNAs of factors involved in proliferation, invasiveness, and inflammation. Disruption of the gene that codes for TTP (Zfp36) led to severe arthritis, autoimmunity, cachexia and dermatitis in mice. It has been shown that these phenotypes were mostly due to excessive TNFα levels in the affected tissues. We have previously reported that TTP expression is required for lactation maintenance. Our results indicated that conditional MG TTP-KO female mice displayed early involution due to the untimely induction of pro-inflammatory pathways led mostly by TNFα overexpression. Here we show that reducing TTP levels not only affects the fully differentiated mammary gland, but also harms morphogenesis of this tissue by impairing the progenitor cell population. We found that Zfp36 expression is linked to mammary stemness in human and mice. In addition, diminishing TTP expression and activity induced apoptosis of stem-like mouse mammary cells, reduced its ability to form mammospheres in culture and to develop into complete glands when implanted into cleared mammary fat pads in vivo. Our results show that survival of the stem-like cells is compromised by increased levels of inflammatory cytokines and stimulation of signaling cascades involving NFκB, STAT3 and MAPK-p38 activation. Moreover, TNFα overexpression and the consequent p38 phosphorylation would be the leading cause of progenitor cell death upon TTP expression restriction. Taken together, our results reveal the relevance of TTP for the maintenance of the mammary progenitor cell compartment by maintaining local TNFα levels at bay.
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Affiliation(s)
- Micaela Stedile
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
| | - Angela Lara Montero
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
| | - Martín Emilio García Solá
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
| | - María Victoria Goddio
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
| | - Inés Beckerman
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
| | - Emilia Bogni
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
| | - Marina Ayre
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
| | - Zaira Naguila
- Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires (UBA), Ciudad de Buenos Aires, Argentina
| | - Omar A. Coso
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
- Departamento de Fisiología, Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires (UBA), Ciudad de Buenos Aires, Argentina
| | - Edith C. Kordon
- Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas (IFIBYNE-UBA-CONICET), Ciudad de Buenos Aires, Argentina
- Departamento de Química Biológica (DQB), Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires (UBA), Ciudad de Buenos Aires, Argentina
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5
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Freen-van Heeren JJ. Posttranscriptional Events Orchestrate Immune Homeostasis of CD8 + T Cells. Methods Mol Biol 2024; 2782:65-80. [PMID: 38622392 DOI: 10.1007/978-1-0716-3754-8_4] [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] [Indexed: 04/17/2024]
Abstract
Maintaining immune homeostasis is instrumental for host health. Immune cells, such as T cells, are instrumental for the eradication of pathogenic bacteria, fungi and viruses. Furthermore, T cells also play a major role in the fight against cancer. Through the formation of immunological memory, a pool of antigen-experienced T cells remains in the body to rapidly protect the host upon reinfection or retransformation. In order to perform their protective function, T cells produce cytolytic molecules, such as granzymes and perforin, and cytokines such as interferon γ and tumor necrosis factor α. Recently, it has become evident that posttranscriptional regulatory events dictate the kinetics and magnitude of cytokine production by murine and human CD8+ T cells. Here, the recent literature regarding the role posttranscriptional regulation plays in maintaining immune homeostasis of antigen-experienced CD8+ T cells is reviewed.
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6
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Liu X, Han W, Hu X. Post-transcriptional regulation of myeloid cell-mediated inflammatory responses. Adv Immunol 2023; 160:59-82. [PMID: 38042586 DOI: 10.1016/bs.ai.2023.09.001] [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] [Indexed: 12/04/2023]
Abstract
Myeloid cells, particularly macrophages, act as the frontline responders to infectious agents and initiate inflammation. While the molecular mechanisms driving inflammatory responses have primarily focused on pattern recognition by myeloid cells and subsequent transcriptional events, it is crucial to note that post-transcriptional regulation plays a pivotal role in this process. In addition to the transcriptional regulation of innate immune responses, additional layers of intricate network of post-transcriptional mechanisms critically determine the quantity and duration of key inflammatory products and thus the outcome of immune responses. A multitude of mechanisms governing post-transcriptional regulation in innate immunity have been uncovered, encompassing RNA alternative splicing, mRNA stability, and translational regulation. This review encapsulates the current insights into the post-transcriptional regulation of inflammatory genes within myeloid cells, with particular emphasis on translational regulation during inflammation. While acknowledging the advancements, we also shed light on the existing gaps in immunological research pertaining to post-transcriptional levels and propose perspectives that controlling post-transcriptional process may serve as potential targets for therapeutic interventions in inflammatory diseases.
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Affiliation(s)
- Xingxian Liu
- Institute for Immunology, Tsinghua University, Beijing, P.R. China; Department of Basic Medical Sciences, Tsinghua University, Beijing, P.R. China; Tsinghua-Peking Center for Life Sciences, Beijing, P.R. China; Beijing Key Laboratory for Immunological Research on Chronic Diseases, Beijing, P.R. China
| | - Weidong Han
- Department of Bio-therapeutic, the First Medical Center, Chinese PLA General Hospital, Beijing, P.R. China
| | - Xiaoyu Hu
- Institute for Immunology, Tsinghua University, Beijing, P.R. China; Department of Basic Medical Sciences, Tsinghua University, Beijing, P.R. China; Tsinghua-Peking Center for Life Sciences, Beijing, P.R. China; The State Key Laboratory of Membrane Biology, Beijing, P.R. China.
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7
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Guha A, Husain MA, Si Y, Nabors LB, Filippova N, Promer G, Smith R, King PH. RNA regulation of inflammatory responses in glia and its potential as a therapeutic target in central nervous system disorders. Glia 2023; 71:485-508. [PMID: 36380708 DOI: 10.1002/glia.24288] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Revised: 09/29/2022] [Accepted: 10/14/2022] [Indexed: 11/17/2022]
Abstract
A major hallmark of neuroinflammation is the activation of microglia and astrocytes with the induction of inflammatory mediators such as IL-1β, TNF-α, iNOS, and IL-6. Neuroinflammation contributes to disease progression in a plethora of neurological disorders ranging from acute CNS trauma to chronic neurodegenerative disease. Posttranscriptional pathways of mRNA stability and translational efficiency are major drivers for the expression of these inflammatory mediators. A common element in this level of regulation centers around the adenine- and uridine-rich element (ARE) which is present in the 3' untranslated region (UTR) of the mRNAs encoding these inflammatory mediators. (ARE)-binding proteins (AUBPs) such as Human antigen R (HuR), Tristetraprolin (TTP) and KH- type splicing regulatory protein (KSRP) are key nodes for directing these posttranscriptional pathways and either promote (HuR) or suppress (TTP and KSRP) glial production of inflammatory mediators. This review will discuss basic concepts of ARE-mediated RNA regulation and its impact on glial-driven neuroinflammatory diseases. We will discuss strategies to target this novel level of gene regulation for therapeutic effect and review exciting preliminary studies that underscore its potential for treating neurological disorders.
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Affiliation(s)
- Abhishek Guha
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Mohammed Amir Husain
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Ying Si
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
- Department Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - L Burt Nabors
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Natalia Filippova
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Grace Promer
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Reed Smith
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Peter H King
- Department Neurology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
- Department Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
- Birmingham Department of Veterans Health Care System, Birmingham, Alabama, USA
- Center for Neurodegeneration and Experimental Therapeutics, The University of Alabama at Birmingham, Birmingham, USA
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Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells. Int J Mol Sci 2022; 23:ijms232415485. [PMID: 36555130 PMCID: PMC9779314 DOI: 10.3390/ijms232415485] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2022] [Revised: 11/28/2022] [Accepted: 12/01/2022] [Indexed: 12/13/2022] Open
Abstract
Compared to transcription initiation, much less is known about transcription termination. In particular, large-scale mutagenesis studies have, so far, primarily concentrated on promoter and enhancer, but not terminator sequences. Here, we used a massively parallel reporter assay (MPRA) to systematically analyze the influence of short (8 bp) sequence variants (mutations) located downstream of the polyadenylation signal (PAS) on the steady-state mRNA level of the upstream gene, employing an eGFP reporter and human HEK293T cells as a model system. In total, we evaluated 227,755 mutations located at different overlapping positions within +17..+56 bp downstream of the PAS for their ability to regulate the reporter gene expression. We found that the positions +17..+44 bp downstream of the PAS are more essential for gene upregulation than those located more distal to the PAS, and that the mutation sequences ensuring high levels of eGFP mRNA expression are extremely T-rich. Next, we validated the positive effect of a couple of mutations identified in the MPRA screening on the eGFP and luciferase protein expression. The most promising mutation increased the expression of the reporter proteins 13-fold and sevenfold on average in HEK293T and CHO cells, respectively. Overall, these findings might be useful for further improving the efficiency of production of therapeutic products, e.g., recombinant antibodies.
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9
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Lai HC, Ho UY, James A, De Souza P, Roberts TL. RNA metabolism and links to inflammatory regulation and disease. Cell Mol Life Sci 2021; 79:21. [PMID: 34971439 PMCID: PMC11072290 DOI: 10.1007/s00018-021-04073-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2021] [Revised: 09/29/2021] [Accepted: 10/22/2021] [Indexed: 11/29/2022]
Abstract
Inflammation is vital to protect the host against foreign organism invasion and cellular damage. It requires tight and concise gene expression for regulation of pro- and anti-inflammatory gene expression in immune cells. Dysregulated immune responses caused by gene mutations and errors in post-transcriptional regulation can lead to chronic inflammatory diseases and cancer. The mechanisms underlying post-transcriptional gene expression regulation include mRNA splicing, mRNA export, mRNA localisation, mRNA stability, RNA/protein interaction, and post-translational events such as protein stability and modification. The majority of studies to date have focused on transcriptional control pathways. However, post-transcriptional regulation of mRNA in eukaryotes is equally important and related information is lacking. In this review, we will focus on the mechanisms involved in the pre-mRNA splicing events, mRNA surveillance, RNA degradation pathways, disorders or symptoms caused by mutations or errors in post-transcriptional regulation during innate immunity especially toll-like receptor mediated pathways.
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Affiliation(s)
- Hui-Chi Lai
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia.
- South West Sydney Clinical School, UNSW Australia, Liverpool, NSW, Australia.
| | - Uda Y Ho
- School of Biomedical Sciences, Faculty of Medicine, University of Queensland, Brisbane, Australia
| | - Alexander James
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
| | - Paul De Souza
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
- School of Medicine, University of Wollongong, Wollongong, NSW, Australia
- School of Medicine, Western Sydney University, Macarthur, NSW, Australia
| | - Tara L Roberts
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
- South West Sydney Clinical School, UNSW Australia, Liverpool, NSW, Australia
- School of Medicine, Western Sydney University, Macarthur, NSW, Australia
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10
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Guo S, Lin S. mRNA alternative polyadenylation (APA) in regulation of gene expression and diseases. Genes Dis 2021; 10:165-174. [PMID: 37013028 PMCID: PMC10066270 DOI: 10.1016/j.gendis.2021.09.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2021] [Revised: 08/26/2021] [Accepted: 09/07/2021] [Indexed: 11/16/2022] Open
Abstract
The mRNA polyadenylation plays essential function in regulation of mRNA metabolism. Mis-regulations of mRNA polyadenylation are frequently linked with aberrant gene expression and disease progression. Under the action of polyadenylate polymerase, poly(A) tail is synthesized after the polyadenylation signal (PAS) sites on the mRNAs. Alternative polyadenylation (APA) often occurs in mRNAs with multiple poly(A) sites, producing different 3' ends for transcript variants, and therefore plays important functions in gene expression regulation. In this review, we first summarize the classical process of mRNA 3'-terminal formation and discuss the length control mechanisms of poly(A) in nucleus and cytoplasm. Then we review the research progress on alternative polyadenylation regulation and the APA site selection mechanism. Finally, we summarize the functional roles of APA in the regulation of gene expression and diseases including cancers.
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Affiliation(s)
- Siyao Guo
- Center for Translational Medicine, Precision Medicine Institute, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China
- State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, China
| | - Shuibin Lin
- Center for Translational Medicine, Precision Medicine Institute, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China
- State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, China
- Corresponding author. Center for Translational Medicine, Precision Medicine Institute, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China.
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Rodríguez-Gómez G, Paredes-Villa A, Cervantes-Badillo MG, Gómez-Sonora JP, Jorge-Pérez JH, Cervantes-Roldán R, León-Del-Río A. Tristetraprolin: A cytosolic regulator of mRNA turnover moonlighting as transcriptional corepressor of gene expression. Mol Genet Metab 2021; 133:137-147. [PMID: 33795191 DOI: 10.1016/j.ymgme.2021.03.015] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2020] [Revised: 03/23/2021] [Accepted: 03/24/2021] [Indexed: 01/12/2023]
Abstract
Tristetraprolin (TTP) is a nucleocytoplasmic 326 amino acid protein whose sequence is characterized by possessing two CCCH-type zinc finger domains. In the cytoplasm TTP function is to promote the degradation of mRNAs that contain adenylate/uridylate-rich elements (AREs). Mechanistically, TTP promotes the recruitment of poly(A)-specific deadenylases and exoribonucleases. By reducing the half-life of about 10% of all the transcripts in the cell TTP has been shown to participate in multiple cell processes that include regulation of gene expression, cell proliferation, metabolic homeostasis and control of inflammation and immune responses. However, beyond its role in mRNA decay, in the cell nucleus TTP acts as a transcriptional coregulator by interacting with chromatin modifying enzymes. TTP has been shown to repress the transactivation of NF-κB and estrogen receptor suggesting the possibility that it participates in the transcriptional regulation of hundreds of genes in human cells and its possible involvement in breast cancer progression. In this review, we discuss the cytoplasmic and nuclear functions of TTP and the effect of the dysregulation of its protein levels in the development of human diseases. We suggest that TTP be classified as a moonlighting tumor supressor protein that regulates gene expression through two different mechanims; the decay of ARE-mRNAs and a transcriptional coregulatory function.
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Affiliation(s)
- Gabriel Rodríguez-Gómez
- Programa de Investigación en Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
| | - Alejandro Paredes-Villa
- Programa de Investigación en Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
| | - Mayte Guadalupe Cervantes-Badillo
- Programa de Investigación en Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
| | - Jessica Paola Gómez-Sonora
- Programa de Investigación en Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
| | - Jesús H Jorge-Pérez
- Programa de Investigación en Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
| | - Rafael Cervantes-Roldán
- Programa de Investigación en Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
| | - Alfonso León-Del-Río
- Programa de Investigación en Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico.
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12
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The mRNA-destabilizing protein Tristetraprolin targets "meiosis arrester" Nppc mRNA in mammalian preovulatory follicles. Proc Natl Acad Sci U S A 2021; 118:2018345118. [PMID: 34031239 DOI: 10.1073/pnas.2018345118] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
C-natriuretic peptide (CNP) and its receptor guanylyl cyclase, natriuretic peptide receptor 2 (NPR2), are key regulators of cyclic guanosine monophosphate (cGMP) homeostasis. The CNP-NPR2-cGMP signaling cascade plays an important role in the progression of oocyte meiosis, which is essential for fertility in female mammals. In preovulatory ovarian follicles, the luteinizing hormone (LH)-induced decrease in CNP and its encoding messenger RNA (mRNA) natriuretic peptide precursor C (Nppc) are a prerequisite for oocyte meiotic resumption. However, it has never been determined how LH decreases CNP/Nppc In the present study, we identified that tristetraprolin (TTP), also known as zinc finger protein 36 (ZFP36), a ubiquitously expressed mRNA-destabilizing protein, is the critical mechanism that underlies the LH-induced decrease in Nppc mRNA. Zfp36 mRNA was transiently up-regulated in mural granulosa cells (MGCs) in response to the LH surge. Loss- and gain-of-function analyses indicated that TTP is required for Nppc mRNA degradation in preovulatory MGCs by targeting the rare noncanonical AU-rich element harbored in the Nppc 3' UTR. Moreover, MGC-specific knockout of Zfp36, as well as lentivirus-mediated knockdown in vivo, impaired the LH/hCG-induced Nppc mRNA decline and oocyte meiotic resumption. Furthermore, we found that LH/hCG activates Zfp36/TTP expression through the EGFR-ERK1/2-dependent pathway. Our findings reveal a functional role of TTP-induced mRNA degradation, a global posttranscriptional regulation mechanism, in orchestrating the progression of oocyte meiosis. We also provided a mechanism for understanding CNP-dependent cGMP homeostasis in diverse cellular processes.
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13
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Freen-van Heeren JJ. Post-transcriptional control of T-cell cytokine production: Implications for cancer therapy. Immunology 2021; 164:57-72. [PMID: 33884612 DOI: 10.1111/imm.13339] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2021] [Revised: 03/22/2021] [Accepted: 03/30/2021] [Indexed: 01/05/2023] Open
Abstract
As part of the adaptive immune system, T cells are vital for the eradication of infected and malignantly transformed cells. To perform their protective function, T cells produce effector molecules that are either directly cytotoxic, such as granzymes, perforin, interferon-γ and tumour necrosis factor α, or attract and stimulate (immune) cells, such as interleukin-2. As these molecules can also induce immunopathology, tight control of their production is required. Indeed, inflammatory cytokine production is regulated on multiple levels. Firstly, locus accessibility and transcription factor availability and activity determine the amount of mRNA produced. Secondly, post-transcriptional mechanisms, influencing mRNA splicing/codon usage, stability, decay, localization and translation rate subsequently determine the amount of protein that is produced. In the immune suppressive environments of tumours, T cells gradually lose the capacity to produce effector molecules, resulting in tumour immune escape. Recently, the role of post-transcriptional regulation in fine-tuning T-cell effector function has become more appreciated. Furthermore, several groups have shown that exhausted or dysfunctional T cells from cancer patients or murine models possess mRNA for inflammatory mediators, but fail to produce effector molecules, hinting that post-transcriptional events also play a role in hampering tumour-infiltrating lymphocyte effector function. Here, the post-transcriptional regulatory events governing T-cell cytokine production are reviewed, with a specific focus on the importance of post-transcriptional regulation in anti-tumour responses. Furthermore, potential approaches to circumvent tumour-mediated dampening of T-cell effector function through the (dis)engagement of post-transcriptional events are explored, such as CRISPR/Cas9-mediated genome editing or chimeric antigen receptors.
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14
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Freen-van Heeren JJ. Using CRISPR to enhance T cell effector function for therapeutic applications. Cytokine X 2021; 3:100049. [PMID: 33604565 PMCID: PMC7885876 DOI: 10.1016/j.cytox.2020.100049] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Accepted: 12/15/2020] [Indexed: 12/12/2022] Open
Abstract
T cells are critical to fight pathogenic microbes and combat malignantly transformed cells in the fight against cancer. To exert their effector function, T cells produce effector molecules, such as the pro-inflammatory cytokines IFN-γ, TNF-α and IL-2. Tumors possess many inhibitory mechanisms that dampen T cell effector function, limiting the secretion of cytotoxic molecules. As a result, the control and elimination of tumors is impaired. Through recent advances in genomic editing, T cells can now be successfully modified via CRISPR/Cas9 technology. For instance, engaging (post-)transcriptional mechanisms to enhance T cell cytokine production, the retargeting of T cell antigen specificity or rendering T cells refractive to inhibitory receptor signaling can augment T cell effector function. Therefore, CRISPR/Cas9-mediated genome editing might provide novel strategies for cancer immunotherapy. Recently, the first-in-patient clinical trial was successfully performed with CRISPR/Cas9-modified human T cell therapy. In this review, a brief overview of currently available techniques is provided, and recent advances in T cell genomic engineering for the enhancement of T cell effector function for therapeutic purposes are discussed.
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Key Words
- AP-1, activator protein 1
- ARE, AU-rich element
- ARE-Del, deletion of the 3′UTR AREs from the Ifng/IFNG gene
- CAR T cells
- CAR, Chimeric Antigen Receptor
- CRISPR
- CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat
- CRS, cytokine release syndrome
- CTLA-4, cytotoxic T-lymphocyte-associated protein 4
- Cas, CRISPR-associated
- Cas9
- Cytokines
- DGK, Diacylglycerol kinase
- DHX37, DEAH-box helicase 37
- EBV, Epstein Barr virus
- FOXP3, Forkhead box P3
- GATA, GATA binding protein
- Genome editing
- IFN, interferon
- IL, interleukin
- LAG-3, Lymphocyte Activating 3
- NF-κB, nuclear factor of activated B cells
- PD-1, Programmed cell Death 1
- PD-L1, Programmed Death Ligand 1
- PTPN2, Protein Tyrosine Phosphatase Non-Receptor 2
- Pdia3, Protein Disulfide Isomerase Family A Member 3
- RBP, RNA-binding protein
- RNP, ribonuclear protein
- T cell effector function
- T cells
- TCR, T cell receptor
- TGF, transforming growth factor
- TIL, Tumor Infiltrating Lymphocyte
- TLRs, Toll-like receptors
- TNF, tumor necrosis factor
- TRAC, TCR-α chain
- TRBC, TCR-β chain
- UTR, untranslated region
- tTCR, transgenic TCR
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15
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Binas O, Tants JN, Peter SA, Janowski R, Davydova E, Braun J, Niessing D, Schwalbe H, Weigand JE, Schlundt A. Structural basis for the recognition of transiently structured AU-rich elements by Roquin. Nucleic Acids Res 2020; 48:7385-7403. [PMID: 32491174 PMCID: PMC7367199 DOI: 10.1093/nar/gkaa465] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2020] [Revised: 05/16/2020] [Accepted: 05/20/2020] [Indexed: 12/26/2022] Open
Abstract
Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3′-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3′-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.
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Affiliation(s)
- Oliver Binas
- Institute for Organic Chemistry and Chemical Biology, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance (BMRZ), 60438 Frankfurt, Germany
| | - Jan-Niklas Tants
- Institute for Molecular Biosciences, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance (BMRZ), 60438 Frankfurt, Germany
| | - Stephen A Peter
- Department of Biology, Technical University of Darmstadt, Darmstadt 64287, Germany
| | - Robert Janowski
- Institute of Structural Biology, Helmholtz-Zentrum München, 85764 Neuherberg, Germany
| | - Elena Davydova
- Institute of Structural Biology, Helmholtz-Zentrum München, 85764 Neuherberg, Germany
| | - Johannes Braun
- Department of Biology, Technical University of Darmstadt, Darmstadt 64287, Germany
| | - Dierk Niessing
- Institute of Structural Biology, Helmholtz-Zentrum München, 85764 Neuherberg, Germany.,Institute of Pharmaceutical Biotechnology, Ulm University, 89081 Ulm, Germany
| | - Harald Schwalbe
- Institute for Organic Chemistry and Chemical Biology, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance (BMRZ), 60438 Frankfurt, Germany
| | - Julia E Weigand
- Department of Biology, Technical University of Darmstadt, Darmstadt 64287, Germany
| | - Andreas Schlundt
- Institute for Molecular Biosciences, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance (BMRZ), 60438 Frankfurt, Germany
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16
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Sung SSJ, Fu SM. A novel immunofluorescence detection method for renal cell-type specific in situ cytokine production by confocal microscopy. MethodsX 2020; 7:100935. [PMID: 32577408 PMCID: PMC7303990 DOI: 10.1016/j.mex.2020.100935] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2019] [Accepted: 05/20/2020] [Indexed: 11/25/2022] Open
Abstract
The detection of cytokines production in tissues is subjected to significant limitations: (1) Cytokine protein production frequently does not correlate with mRNA levels. (2) Cytokines are secreted rapidly and dissipate from the cellular source, thus making detection difficult. (3) The synthetic rate of many cytokines are low. (4) Tissue fixation ablates antigenic sites and diminishes detection signals. The identification of the cellular sources of cytokines poses an additional challenge because of the lack of suitable and readily available cellular markers. In our renal cytokine production studies in lupus nephritis, we have established methods to resolve problems associated with the identification of cellular sources of pertinent cytokines in the glomerulus and interstitium. Four-color confocal microscopy was used to colocalize cell-type specific markers with cytokines. The cytokine signal was amplified by the incubation of tissue slices in medium containing pan-specific stimulants plus secretion blockers. Tissue fixation was optimized to provide sharp crisp signals. Commercially available Ab suitable for fluorochrome labeling were used to establish cell-specific markers in the tubules and glomeruli. This combination of optimizations allowed us to define the cellular sources of important glomerular cytokines including TNF-α, IL-6, and IL-1β which appear to form a cytokine circuit in glomerulonephritis pathogenesis. ● Tissue stimulation and secretion blocking for cytokine detection ● Fixation optimization and Ab source identification for direct staining ● Colocalization of cytokines and renal cell-type specific markers.
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Affiliation(s)
- Sun-Sang J Sung
- Center for Immunity, Inflammation, and Regenerative Medicine
| | - Shu Man Fu
- Center for Immunity, Inflammation, and Regenerative Medicine.,Division of Rheumatology, Department of Medicine, Charlottesville, VA 22908
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17
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Freen-van Heeren JJ, Popović B, Guislain A, Wolkers MC. Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production. Eur J Immunol 2020; 50:949-958. [PMID: 32112565 PMCID: PMC7384093 DOI: 10.1002/eji.201948458] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Revised: 01/27/2020] [Accepted: 02/27/2020] [Indexed: 12/25/2022]
Abstract
Long‐lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro‐inflammatory cytokine IFN‐γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN‐γ is tightly regulated through adenylate uridylate–rich elements (AREs) that are located in the 3′ untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti‐tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR‐Cas9 technology, we deleted the ARE region from the IFNG 3′ UTR in peripheral blood‐derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN‐γ protein‐producing T cells. Importantly, combining MART‐1 T cell receptor engineering with ARE‐Del gene editing showed that this was also true for antigen‐specific activation of T cells. MART‐1‐specific ARE‐Del T cells showed higher percentages of IFN‐γ producing T cells in response to MART‐1 expressing tumor cells. Combined, our study reveals that ARE‐mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen‐specific ARE‐Del T cells is feasible, a feature that could potentially be used for therapeutical purposes.
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Affiliation(s)
- Julian J Freen-van Heeren
- Department of Hematopoiesis, Sanquin Research-Amsterdam UMC Landsteiner Laboratory, Amsterdam, The Netherlands.,Department of Hematopoiesis, Oncode Institute, Amsterdam, The Netherlands
| | - Branka Popović
- Department of Hematopoiesis, Sanquin Research-Amsterdam UMC Landsteiner Laboratory, Amsterdam, The Netherlands.,Department of Hematopoiesis, Oncode Institute, Amsterdam, The Netherlands
| | - Aurélie Guislain
- Department of Hematopoiesis, Sanquin Research-Amsterdam UMC Landsteiner Laboratory, Amsterdam, The Netherlands.,Department of Hematopoiesis, Oncode Institute, Amsterdam, The Netherlands
| | - Monika C Wolkers
- Department of Hematopoiesis, Sanquin Research-Amsterdam UMC Landsteiner Laboratory, Amsterdam, The Netherlands.,Department of Hematopoiesis, Oncode Institute, Amsterdam, The Netherlands
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18
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Sung SSJ, Fu SM. Interactions among glomerulus infiltrating macrophages and intrinsic cells via cytokines in chronic lupus glomerulonephritis. J Autoimmun 2019; 106:102331. [PMID: 31495649 DOI: 10.1016/j.jaut.2019.102331] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Revised: 08/23/2019] [Accepted: 08/25/2019] [Indexed: 10/26/2022]
Abstract
Inflammation plays a key role in the pathogenesis of lupus nephritis (LN) and inflammatory cytokines within the glomeruli are critical in this process. However, little information is available for the identities of the cell types that are primarily responsible for the production and function of the various cytokines. We have devised a novel method to visualize cytokine signals in the kidney by confocal microscopy and found that cytokine production within the glomerulus is cell type-specific and under translational control. In the lupus-prone NZM2328 mice with chronic glomerulonephritis, IL-6, IL-1β, and TNF-α in the glomerulus were produced predominantly by mesangial cells, podocytes, and glomerulus-infiltrating blood-derived macrophages, respectively. Microarray and RNASeq analyses showed that these cells expressed the receptors for these cytokines. Together the 3 cell types form a cytokine circuit in amplifying cytokine responses in LN. The intrinsic cells and infiltrating macrophages also produced other cytokines including M-CSF, SCF, and IL-34 that constituted within the enclosed glomerular space the soluble effector milieu which may mediate cellular damage and proliferation, and cytokine transcriptional and translation regulation. IL-10 and IL-1β were translationally regulated in the glomeruli in the intact kidney in a cell type-specific manner. The production of these 2 cytokines by infiltrating macrophages was undetectable in a visualization system for in situ protein accumulation despite high mRNA expression levels. However, these macrophages in isolated glomeruli which are released from Bowman's capsules produced large amounts of IL-10 and IL-1β. These data reveal the complexity of cytokine regulation, production, and function in the glomerulus and provide a model in which cytokine blocking may be beneficial in LN treatment.
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Affiliation(s)
- Sun-Sang J Sung
- Center for Immunity, Inflammation, and Regenerative Medicine, Departments of Medicine, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
| | - Shu Man Fu
- Center for Immunity, Inflammation, and Regenerative Medicine, Departments of Medicine, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA; Division of Rheumatology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
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19
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Legrand N, Dixon DA, Sobolewski C. AU-rich element-binding proteins in colorectal cancer. World J Gastrointest Oncol 2019; 11:71-90. [PMID: 30788036 PMCID: PMC6379757 DOI: 10.4251/wjgo.v11.i2.71] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/05/2018] [Revised: 12/11/2018] [Accepted: 01/01/2019] [Indexed: 02/05/2023] Open
Abstract
Trans-acting factors controlling mRNA fate are critical for the post-transcriptional regulation of inflammation-related genes, as well as for oncogene and tumor suppressor expression in human cancers. Among them, a group of RNA-binding proteins called “Adenylate-Uridylate-rich elements binding proteins” (AUBPs) control mRNA stability or translation through their binding to AU-rich elements enriched in the 3’UTRs of inflammation- and cancer-associated mRNA transcripts. AUBPs play a central role in the recruitment of target mRNAs into small cytoplasmic foci called Processing-bodies and stress granules (also known as P-body/SG). Alterations in the expression and activities of AUBPs and P-body/SG assembly have been observed to occur with colorectal cancer (CRC) progression, indicating the significant role AUBP-dependent post-transcriptional regulation plays in controlling gene expression during CRC tumorigenesis. Accordingly, these alterations contribute to the pathological expression of many early-response genes involved in prostaglandin biosynthesis and inflammation, along with key oncogenic pathways. In this review, we summarize the current role of these proteins in CRC development. CRC remains a major cause of cancer mortality worldwide and, therefore, targeting these AUBPs to restore efficient post-transcriptional regulation of gene expression may represent an appealing therapeutic strategy.
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Affiliation(s)
- Noémie Legrand
- Department of Microbiology, Faculty of Medicine, University of Geneva, Geneva CH-1211, Switzerland
| | - Dan A Dixon
- Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, and University of Kansas Cancer Center, Kansas City, KS 66045, United States
| | - Cyril Sobolewski
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva CH-1211, Switzerland
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20
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Translational repression of pre-formed cytokine-encoding mRNA prevents chronic activation of memory T cells. Nat Immunol 2018; 19:828-837. [PMID: 29988089 DOI: 10.1038/s41590-018-0155-6] [Citation(s) in RCA: 69] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2014] [Accepted: 05/22/2018] [Indexed: 02/05/2023]
Abstract
Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.
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21
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Blanco FF, Preet R, Aguado A, Vishwakarma V, Stevens LE, Vyas A, Padhye S, Xu L, Weir SJ, Anant S, Meisner-Kober N, Brody JR, Dixon DA. Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis. Oncotarget 2018; 7:74043-74058. [PMID: 27677075 PMCID: PMC5342034 DOI: 10.18632/oncotarget.12189] [Citation(s) in RCA: 87] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2016] [Accepted: 08/11/2016] [Indexed: 12/12/2022] Open
Abstract
Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality. Observed during CRC tumorigenesis is loss of post-transcriptional regulation of tumor-promoting genes such as COX-2, TNFα and VEGF. Overexpression of the RNA-binding protein HuR (ELAVL1) occurs during colon tumorigenesis and is abnormally present within the cytoplasm, where it post-transcriptionally regulates genes through its interaction with 3′UTR AU-rich elements (AREs). Here, we examine the therapeutic potential of targeting HuR using MS-444, a small molecule HuR inhibitor. Treatment of CRC cells with MS-444 resulted in growth inhibition and increased apoptotic gene expression, while similar treatment doses in non-transformed intestinal cells had no appreciable effects. Mechanistically, MS-444 disrupted HuR cytoplasmic trafficking and released ARE-mRNAs for localization to P-bodies, but did not affect total HuR expression levels. This resulted in MS-444-mediated inhibition of COX-2 and other ARE-mRNA expression levels. Importantly, MS-444 was well tolerated and inhibited xenograft CRC tumor growth through enhanced apoptosis and decreased angiogenesis upon intraperitoneal administration. In vivo treatment of MS-444 inhibited HuR cytoplasmic localization and decreased COX-2 expression in tumors. These findings provide evidence that therapeutic strategies to target HuR in CRC warrant further investigation in an effort to move this approach to the clinic.
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Affiliation(s)
- Fernando F Blanco
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA.,Department of Surgery, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
| | - Ranjan Preet
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA
| | - Andrea Aguado
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA
| | - Vikalp Vishwakarma
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA
| | - Laura E Stevens
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA
| | - Alok Vyas
- Maharashtra Cosmopolitan Education Society's ISTRA, Azam Campus, University of Pune, India
| | - Subhash Padhye
- Maharashtra Cosmopolitan Education Society's ISTRA, Azam Campus, University of Pune, India
| | - Liang Xu
- University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA.,Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
| | - Scott J Weir
- Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS, USA.,University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
| | - Shrikant Anant
- Department of Surgery, University of Kansas Medical Center, Kansas City, KS, USA.,University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
| | | | - Jonathan R Brody
- Department of Surgery, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
| | - Dan A Dixon
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA.,University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
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22
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Guo L, Louis IVS, Bohjanen PR. Post-transcriptional regulation of cytokine expression and signaling. CURRENT TRENDS IN IMMUNOLOGY 2018; 19:33-40. [PMID: 30568341 PMCID: PMC6296478] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Cytokines and cytokine signaling pathways are crucial for regulating cellular functions, including cell growth, proliferation, differentiation, and cell death. Cytokines regulate physiological processes such as immune responses and maintain immune homeostasis, and they also mediate pathological conditions such as autoimmune diseases and cancer. Hence, the precise control of the expression of cytokines and the transduction of cytokine signals is tightly regulated at transcriptional and post-transcriptional levels. In particular, post-transcriptional regulation at the level of mRNA stability is critical for coordinating cytokine expression and cytokine signaling. Numerous cytokine transcripts contain AU-rich elements (AREs), whereas transcripts encoding numerous components of cytokine signaling pathways contain GU-rich elements (GREs). AREs and GREs are mRNA decay elements that mediate rapid mRNA degradation. Through ARE- and GRE-mediated decay mechanisms, immune cells selectively and specifically regulate cytokine networks during immune responses. Aberrant expression and stability of ARE- or GRE-containing transcripts that encode cytokines or components of cytokine signaling pathways are observed in disease states, including cancer. In this review, we focus on the role of AREs and GREs in regulating cytokine expression and signal transduction at the level of mRNA stability.
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Affiliation(s)
- Liang Guo
- Department of Medicine, Division of Infectious Diseases and International Medicine, Program in Infection and Immunity, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology Training Program, University of Minnesota, Minneapolis, MN 55455, USA
- Graduate Program in Comparative and Molecular Bioscience, University of Minnesota, Minneapolis, MN 55455, USA
| | - Irina Vlasova-St. Louis
- Department of Medicine, Division of Infectious Diseases and International Medicine, Program in Infection and Immunity, University of Minnesota, Minneapolis, MN 55455, USA
| | - Paul R. Bohjanen
- Department of Medicine, Division of Infectious Diseases and International Medicine, Program in Infection and Immunity, University of Minnesota, Minneapolis, MN 55455, USA
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology Training Program, University of Minnesota, Minneapolis, MN 55455, USA
- Graduate Program in Comparative and Molecular Bioscience, University of Minnesota, Minneapolis, MN 55455, USA
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Neve J, Patel R, Wang Z, Louey A, Furger AM. Cleavage and polyadenylation: Ending the message expands gene regulation. RNA Biol 2017; 14:865-890. [PMID: 28453393 PMCID: PMC5546720 DOI: 10.1080/15476286.2017.1306171] [Citation(s) in RCA: 93] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2017] [Revised: 03/02/2017] [Accepted: 03/09/2017] [Indexed: 12/13/2022] Open
Abstract
Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3'end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease.
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Affiliation(s)
- Jonathan Neve
- Department of Biochemistry, University of Oxford, Oxford, United Kingdom
| | - Radhika Patel
- Department of Biochemistry, University of Oxford, Oxford, United Kingdom
| | - Zhiqiao Wang
- Department of Biochemistry, University of Oxford, Oxford, United Kingdom
| | - Alastair Louey
- Department of Biochemistry, University of Oxford, Oxford, United Kingdom
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Kumagai Y, Vandenbon A, Teraguchi S, Akira S, Suzuki Y. Genome-wide map of RNA degradation kinetics patterns in dendritic cells after LPS stimulation facilitates identification of primary sequence and secondary structure motifs in mRNAs. BMC Genomics 2016; 17:1032. [PMID: 28155712 PMCID: PMC5259865 DOI: 10.1186/s12864-016-3325-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Immune cells have to change their gene expression patterns dynamically in response to external stimuli such as lipopolysaccharide (LPS). The gene expression is regulated at multiple steps in eukaryotic cells, in which control of RNA levels at both the transcriptional level and the post-transcriptional level plays important role. Impairment of the control leads to aberrant immune responses such as excessive or impaired production of cytokines. However, genome-wide studies focusing on the post-transcriptional control were relatively rare until recently. Moreover, several RNA cis elements and RNA-binding proteins have been found to be involved in the process, but our general understanding remains poor, partly because identification of regulatory RNA motifs is very challenging in spite of its importance. We took advantage of genome-wide measurement of RNA degradation in combination with estimation of degradation kinetics by qualitative approach, and performed de novo prediction of RNA sequence and structure motifs. METHODS To classify genes by their RNA degradation kinetics, we first measured RNA degradation time course in mouse dendritic cells after LPS stimulation and the time courses were clustered to estimate degradation kinetics and to find patterns in the kinetics. Then genes were clustered by their similarity in degradation kinetics patterns. The 3' UTR sequences of a cluster was subjected to de novo sequence or structure motif prediction. RESULTS The quick degradation kinetics was found to be strongly associated with lower gene expression level, immediate regulation (both induction and repression) of gene expression level, and longer 3' UTR length. De novo sequence motif prediction found AU-rich element-like and TTP-binding sequence-like motifs which are enriched in quickly degrading genes. De novo structure motif prediction found a known functional motif, namely stem-loop structure containing sequence bound by RNA-binding protein Roquin and Regnase-1, as well as unknown motifs. CONCLUSIONS The current study indicated that degradation kinetics patterns lead to classification different from that by gene expression and the differential classification facilitates identification of functional motifs. Identification of novel motif candidates implied post-transcriptional controls different from that by known pairs of RNA-binding protein and RNA motif.
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Affiliation(s)
- Yutaro Kumagai
- Quantitative Immunology Research Unit, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
| | - Alexis Vandenbon
- Immuno-Genomics Research Unit, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan
| | - Shunsuke Teraguchi
- Quantitative Immunology Research Unit, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
| | - Shizuo Akira
- Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan
| | - Yutaka Suzuki
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, 277-8561, Japan
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25
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Diverse Strategies Used by Picornaviruses to Escape Host RNA Decay Pathways. Viruses 2016; 8:v8120335. [PMID: 27999393 PMCID: PMC5192396 DOI: 10.3390/v8120335] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2016] [Revised: 12/07/2016] [Accepted: 12/09/2016] [Indexed: 12/24/2022] Open
Abstract
To successfully replicate, viruses protect their genomic material from degradation by the host cell. RNA viruses must contend with numerous destabilizing host cell processes including mRNA decay pathways and viral RNA (vRNA) degradation resulting from the antiviral response. Members of the Picornaviridae family of small RNA viruses have evolved numerous diverse strategies to evade RNA decay, including incorporation of stabilizing elements into vRNA and re-purposing host stability factors. Viral proteins are deployed to disrupt and inhibit components of the decay machinery and to redirect decay machinery to the advantage of the virus. This review summarizes documented interactions of picornaviruses with cellular RNA decay pathways and processes.
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26
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Vlasova-St Louis I, Bohjanen PR. Post-transcriptional regulation of cytokine and growth factor signaling in cancer. Cytokine Growth Factor Rev 2016; 33:83-93. [PMID: 27956133 DOI: 10.1016/j.cytogfr.2016.11.004] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2016] [Accepted: 11/28/2016] [Indexed: 12/11/2022]
Abstract
Cytokines and growth factors regulate cell proliferation, differentiation, migration and apoptosis, and play important roles in coordinating growth signal responses during development. The expression of cytokine genes and the signals transmitted through cytokine receptors are tightly regulated at several levels, including transcriptional and post-transcriptional levels. A majority of cytokine mRNAs, including growth factor transcripts, contain AU-rich elements (AREs) in their 3' untranslated regions that control gene expression by regulating mRNA degradation and changing translational rates. In addition, numerous proteins involved in transmitting signals downstream of cytokine receptors are regulated at the level of mRNA degradation by GU-rich elements (GREs) found in their 3' untranslated regions. Abnormal stabilization and overexpression of ARE or GRE-containing transcripts had been observed in many malignancies, which is a consequence of the malfunction of RNA-binding proteins. In this review, we briefly summarize the role of AREs and GREs in regulating mRNA turnover to coordinate cytokine and growth factor expression, and we describe how dysregulation of mRNA degradation mechanisms contributes to the development and progression of cancer.
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Affiliation(s)
| | - Paul R Bohjanen
- Department of Medicine, University of Minnesota, Minneapolis, MN, USA; Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, USA
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27
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Monosodium urate crystal-induced pro-interleukin-1β production is post-transcriptionally regulated via the p38 signaling pathway in human monocytes. Sci Rep 2016; 6:34533. [PMID: 27694988 PMCID: PMC5046103 DOI: 10.1038/srep34533] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2016] [Accepted: 09/15/2016] [Indexed: 12/23/2022] Open
Abstract
IL-1β is a key mediator of sterile inflammation in response to endogenous particulates, a type of damage-associated molecular pattern (DAMPs) molecule derived from damaged cells. Despite the well-known role of sterile particulates such as monosodium urate (MSU) crystals as inflammasome inducers in monocytes/macrophages, little is known regarding how pro-IL-1β synthesis is induced under sterile inflammatory conditions. We provide evidence that MSU crystals post-transcriptionally induce the rapid production of pro-IL-1β in human primary monocytes. Metabolic labeling and pull-down assays for newly-synthesized proteins clearly showed that MSU crystals rapidly, within 30 min, induce the synthesis of pro-IL-1β as well as global proteins. Notably, MSU crystal-induced pro-IL-1β synthesis is selectively dependent on the p38 MAPK pathway, whereas global protein synthesis is mediated via the mTOR, ERK1/2, and p38 pathways. Furthermore, inhibition of Mnk1, a substrate of p38, blocked MSU crystal-induced pro-IL-1β synthesis downstream of eIF4E phosphorylation. In addition, the p38 MAPK pathway leading to phosphorylation of MK2 was also critical for stabilization of pro-IL-1β mRNA following MSU stimulation. Our findings demonstrate that post-transcriptional regulation via p38 MAPK plays a central role in the rapid synthesis of pro-IL-1β in response to MSU crystals, which is an essential step for IL-1β production in human monocytes.
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28
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Liepelt A, Naarmann-de Vries IS, Simons N, Eichelbaum K, Föhr S, Archer SK, Castello A, Usadel B, Krijgsveld J, Preiss T, Marx G, Hentze MW, Ostareck DH, Ostareck-Lederer A. Identification of RNA-binding Proteins in Macrophages by Interactome Capture. Mol Cell Proteomics 2016; 15:2699-714. [PMID: 27281784 DOI: 10.1074/mcp.m115.056564] [Citation(s) in RCA: 65] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Indexed: 12/11/2022] Open
Abstract
Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA-binding proteins (RBPs) in the post-transcriptional control of the innate immune response. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific LYN substrate 1 (HCLS1 or HS1), a hematopoietic cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.
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Affiliation(s)
- Anke Liepelt
- From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany
| | - Isabel S Naarmann-de Vries
- From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany
| | - Nadine Simons
- From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany
| | - Katrin Eichelbaum
- §European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany
| | - Sophia Föhr
- §European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany
| | - Stuart K Archer
- ¶EMBL-Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Garran Rd, Acton (Canberra) ACT 2601, Australia
| | - Alfredo Castello
- §European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany
| | - Björn Usadel
- ‖Institute of Biology I, RWTH Aachen, Worringer Weg 2, 52074 Aachen, Germany
| | - Jeroen Krijgsveld
- §European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany
| | - Thomas Preiss
- ¶EMBL-Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Garran Rd, Acton (Canberra) ACT 2601, Australia; **Victor Chang Cardiac Research Institute, Darlinghurst (Sydney), New South Wales 2010, Australia
| | - Gernot Marx
- From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany
| | - Matthias W Hentze
- §European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany
| | - Dirk H Ostareck
- From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany;
| | - Antje Ostareck-Lederer
- From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany;
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Dash S, Siddam AD, Barnum CE, Janga SC, Lachke SA. RNA-binding proteins in eye development and disease: implication of conserved RNA granule components. WILEY INTERDISCIPLINARY REVIEWS-RNA 2016; 7:527-57. [PMID: 27133484 DOI: 10.1002/wrna.1355] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/02/2016] [Accepted: 03/21/2016] [Indexed: 01/16/2023]
Abstract
The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1, and Bmp4 are commonly required for their development. In contrast, our understanding of posttranscriptional regulation in eye development and disease, particularly regarding the function of RNA-binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila as well as several vertebrate models such as fish, frog, chicken, and mouse. Interestingly, of the 42 RBPs that have been investigated for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as processing bodies, stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate posttranscriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2, and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly, and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving posttranscriptional regulatory networks in eye development and disease. WIREs RNA 2016, 7:527-557. doi: 10.1002/wrna.1355 For further resources related to this article, please visit the WIREs website.
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Affiliation(s)
- Soma Dash
- Department of Biological Sciences, University of Delaware, Newark, DE, USA
| | - Archana D Siddam
- Department of Biological Sciences, University of Delaware, Newark, DE, USA
| | - Carrie E Barnum
- Department of Biological Sciences, University of Delaware, Newark, DE, USA
| | - Sarath Chandra Janga
- Department of Biohealth Informatics, School of Informatics and Computing, Indiana University & Purdue University Indianapolis, Indianapolis, IN, USA.,Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Salil A Lachke
- Department of Biological Sciences, University of Delaware, Newark, DE, USA.,Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE, USA
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Mattijssen S, Maraia RJ. LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner. Mol Cell Biol 2016; 36:574-84. [PMID: 26644407 PMCID: PMC4751689 DOI: 10.1128/mcb.00804-15] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Revised: 09/14/2015] [Accepted: 11/20/2015] [Indexed: 01/09/2023] Open
Abstract
LARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3' untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of β-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-α), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-α-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-α-TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway.
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Affiliation(s)
- Sandy Mattijssen
- Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
| | - Richard J Maraia
- Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA Commissioned Corps, U.S. Public Health Service, Washington, DC, USA
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31
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Vlasova-St Louis I, Bohjanen PR. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs. Cells 2016; 5:cells5010004. [PMID: 26821046 PMCID: PMC4810089 DOI: 10.3390/cells5010004] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2015] [Revised: 01/20/2016] [Accepted: 01/20/2016] [Indexed: 12/18/2022] Open
Abstract
In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP.
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Affiliation(s)
- Irina Vlasova-St Louis
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.
- Department of Microbiology, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Paul R Bohjanen
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.
- Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, MN 55455, USA.
- Department of Microbiology, University of Minnesota, Minneapolis, MN 55455, USA.
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32
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White MR, Garcin ED. The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein. WILEY INTERDISCIPLINARY REVIEWS-RNA 2015; 7:53-70. [PMID: 26564736 DOI: 10.1002/wrna.1315] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Revised: 09/30/2015] [Accepted: 10/01/2015] [Indexed: 01/26/2023]
Abstract
The glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA (tRNA), the regulation of cellular messenger RNA (mRNA) stability and translation, as well as the regulation of replication and gene expression of many single-stranded RNA viruses. A growing body of evidence supports GAPDH-RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA-binding sequences. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function. WIREs RNA 2016, 7:53-70. doi: 10.1002/wrna.1315 For further resources related to this article, please visit the WIREs website.
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Affiliation(s)
- Michael R White
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, USA
| | - Elsa D Garcin
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, USA
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Bohjanen PR, Moua ML, Guo L, Taye A, Vlasova-St Louis IA. Altered CELF1 binding to target transcripts in malignant T cells. RNA (NEW YORK, N.Y.) 2015; 21:1757-1769. [PMID: 26249002 PMCID: PMC4574752 DOI: 10.1261/rna.049940.115] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/19/2015] [Accepted: 06/29/2015] [Indexed: 06/04/2023]
Abstract
The RNA-binding protein, CELF1, binds to a regulatory sequence known as the GU-rich element (GRE) and controls a network of mRNA transcripts that regulate cellular activation, proliferation, and apoptosis. We performed immunoprecipitation using an anti-CELF1 antibody, followed by identification of copurified transcripts using microarrays. We found that CELF1 is bound to a distinct set of target transcripts in the H9 and Jurkat malignant T-cell lines, compared with primary human T cells. CELF1 was not phosphorylated in resting normal T cells, but in malignant T cells, phosphorylation of CELF1 correlated with its inability to bind to GRE-containing mRNAs that served as CELF1 targets in normal T cells. Lack of binding by CELF1 to these mRNAs in malignant T cells correlated with stabilization and increased expression of these transcripts. Several of these GRE-containing transcripts that encode regulators of cell growth were also stabilized and up-regulated in primary tumor cells from patients with T-cell acute lymphoblastic leukemia. Interestingly, transcripts encoding numerous suppressors of cell proliferation that served as targets of CELF1 in malignant T cells, but not normal T cells, exhibited accelerated degradation and reduced expression in malignant compared with normal T cells, consistent with the known function of CELF1 to mediate degradation of bound transcripts. Overall, CELF1 dysfunction in malignant T cells led to the up-regulation of a subset of GRE-containing transcripts that promote cell growth and down-regulation of another subset that suppress cell growth, producing a net effect that would drive a malignant phenotype.
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Affiliation(s)
- Paul R Bohjanen
- Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, USA Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota 55455, USA
| | - Mai Lee Moua
- Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota 55455, USA
| | - Liang Guo
- Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota 55455, USA
| | - Ammanuel Taye
- Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota 55455, USA
| | - Irina A Vlasova-St Louis
- Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota 55455, USA
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Vlasova-St Louis I, Bohjanen PR. Post-transcriptional regulation of cytokine signaling by AU-rich and GU-rich elements. J Interferon Cytokine Res 2015; 34:233-41. [PMID: 24697201 DOI: 10.1089/jir.2013.0108] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Cytokines are necessary for cell communication to enable responses to external stimuli that are imperative for the survival and maintenance of homeostasis. Dysfunction of the cytokine network has detrimental effects on intra- and extracellular environments. Thus, it is critical that the expression of cytokines and the signals transmitted by cytokines to target cells are tightly regulated at numerous levels, including transcriptional and post-transcriptional levels. Here, we briefly summarize the role of AU-rich elements (AREs) in the regulation of cytokine gene expression at the post-transcriptional level and describe a role for GU-rich elements (GREs) in coordinating the regulation of cytokine signaling. GREs function as post-transcriptional regulators of proteins that control cellular activation, growth, and apoptosis. GREs and AREs work in concert to coordinate cytokine signal transduction pathways. The precise regulation of cytokine signaling is particularly important, because its dysregulation can lead to human diseases.
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Curinha A, Oliveira Braz S, Pereira-Castro I, Cruz A, Moreira A. Implications of polyadenylation in health and disease. Nucleus 2014; 5:508-19. [PMID: 25484187 DOI: 10.4161/nucl.36360] [Citation(s) in RCA: 103] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Polyadenylation is the RNA processing step that completes the maturation of nearly all eukaryotic mRNAs. It is a two-step nuclear process that involves an endonucleolytic cleavage of the pre-mRNA at the 3'-end and the polymerization of a polyadenosine (polyA) tail, which is fundamental for mRNA stability, nuclear export and efficient translation during development. The core molecular machinery responsible for the definition of a polyA site includes several recognition, cleavage and polyadenylation factors that identify and act on a given polyA signal present in a pre-mRNA, usually an AAUAAA hexamer or similar sequence. This mechanism is tightly regulated by other cis-acting elements and trans-acting factors, and its misregulation can cause inefficient gene expression and may ultimately lead to disease. The majority of genes generate multiple mRNAs as a result of alternative polyadenylation in the 3'-untranslated region. The variable lengths of the 3' untranslated regions created by alternative polyadenylation are a recognizable target for differential regulation and clearly affect the fate of the transcript, ultimately modulating the expression of the gene. Over the past few years, several studies have highlighted the importance of polyadenylation and alternative polyadenylation in gene expression and their impact in a variety of physiological conditions, as well as in several illnesses. Abnormalities in the 3'-end processing mechanisms thus represent a common feature among many oncological, immunological, neurological and hematological disorders, but slight imbalances can lead to the natural establishment of a specific cellular state. This review addresses the key steps of polyadenylation and alternative polyadenylation in different cellular conditions and diseases focusing on the molecular effectors that ensure a faultless pre-mRNA 3' end formation.
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Key Words
- 3′ untranslated region
- 3′READS, 3′ Region Extraction and Deep Sequencing
- AD, Alzheimer disease
- APA, Alternative polyadenylation
- AREs, Au-rich elements
- BPV, bovine papilloma virus
- CAH, congenital adrenal hyperplasia
- CFIm25, Cleavage Factor Im 25 kDa
- COX-2, cyclooxygenase 2
- CPSF, Cleavage and Polyadenylation Specificity Factor
- CSTF2, cleavage stimulatory factor-64kDa
- DMKN, dermokine
- DSE, downstream sequence element
- ESC, embryonic stem cells
- FMR1, Fragil X mental retardation 1
- FOXP3, forkhead box P3
- FXPOI, fragile X-associated immature ovarian insufficiency
- FXS, Fragile X syndrome
- FXTAS, fragile X-associated tremor/ataxia syndrome
- HGRG-14, high-glucose-regulated gene
- IMP-1, Insulin-like growth factor 2 mRNA binding protein 1
- IPEX, immune dysfunction, polyendocrinopathy, enteropathy, X-linked
- LPS, lipopolysaccharide
- OPMD, oculopharyngeal muscular dystrophy
- PABPN1, poly(A) binding protein
- PAP, polyA polymerase
- PAS, polyA site
- PD, Parkinson disease
- PDXK, pyridoxal kinase
- PPIE, peptidylpropylisomerase E
- RBP, RNA-binding protein
- RNA Pol II, RNA polymerase II
- SLE, systemic lupus erythematosus
- SMA, Spinal Muscular Atrophy
- SMN, Survival Motor Neuron
- SNP, single nucleotide polymorphism
- StAR, steroigogenic acute regulatory
- TCF/LEF, T cell factor/lymphoid enhancer factor.
- TCF7L2, transcription factor 7-like 2
- TCR, T cell receptor
- TLI, tandem UTR length index
- TNF-α, tumor necrosis factor-α
- USE, upstream sequence element
- UTR, untranslated region
- WAS, Wiskott-Aldrich syndrome
- WASP, Wiskott-Aldrich syndrome protein
- aSyn, α-Synuclein
- aSynL, longest aSyn isoform
- alternative polyadenylation
- cell state
- disease
- gene expression
- miRNA, microRNA
- nuclear 1
- pA signal, polyA signal
- pA tail, polyA tail
- polyadenylation
- siRNAs, small interfering RNAs
- snRNPs, spliceosomal small nuclear ribonucleoproteins
- α-GalA, α-galactosidase A
- μ, IgM heavy-chain mRNA
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Affiliation(s)
- Ana Curinha
- a Gene Regulation Group; IBMC-Instituto de Biologia Molecular e Celular ; Universidade do Porto ; Porto , Portugal
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Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ. Oncogene 2014; 33:4407-17. [PMID: 24469038 PMCID: PMC4150987 DOI: 10.1038/onc.2013.594] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Revised: 10/31/2013] [Accepted: 12/16/2013] [Indexed: 01/29/2023]
Abstract
Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels.
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37
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Carpenter S, Ricci EP, Mercier BC, Moore MJ, Fitzgerald KA. Post-transcriptional regulation of gene expression in innate immunity. Nat Rev Immunol 2014; 14:361-76. [PMID: 24854588 DOI: 10.1038/nri3682] [Citation(s) in RCA: 283] [Impact Index Per Article: 25.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Innate immune responses combat infectious microorganisms by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. Multiple genes within each of these functional categories are coordinately and temporally regulated in response to distinct external stimuli. The substantial potential of these responses to drive pathological inflammation and tissue damage highlights the need for rigorous control of these responses. Although transcriptional control of inflammatory gene expression has been studied extensively, the importance of post-transcriptional regulation of these processes is less well defined. In this Review, we discuss the regulatory mechanisms that occur at the level of mRNA splicing, mRNA polyadenylation, mRNA stability and protein translation, and that have instrumental roles in controlling both the magnitude and duration of the inflammatory response.
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Affiliation(s)
- Susan Carpenter
- 1] Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. [2]
| | - Emiliano P Ricci
- 1] Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. [2]
| | - Blandine C Mercier
- 1] Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. [2]
| | - Melissa J Moore
- Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
| | - Katherine A Fitzgerald
- 1] Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. [2] Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway
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38
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The ROQ domain of Roquin recognizes mRNA constitutive-decay element and double-stranded RNA. Nat Struct Mol Biol 2014; 21:679-85. [PMID: 25026078 PMCID: PMC4125485 DOI: 10.1038/nsmb.2857] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2014] [Accepted: 06/13/2014] [Indexed: 01/03/2023]
Abstract
A conserved stem-loop motif of the constitutive decay element (CDE) in the 3' UTR of mRNAs is recognized by the ROQ domain of Roquin, which mediates mRNA degradation. Here we report two crystal structures of the Homo sapiens ROQ domain in complex with CDE RNA. The ROQ domain has an elongated shape with three subdomains. The 19-nt Hmgxb3 CDE is bound as a stem-loop to domain III. The 23-nt TNF RNA is bound as a duplex to a separate site at the interface between domains I and II. Mutagenesis studies confirm that the ROQ domain has two separate RNA-binding sites, one for stem-loop RNA (A site) and the other for double-stranded RNA (B site). Mutation in either site perturbs the Roquin-mediated degradation of HMGXB3 and IL6 mRNAs in human cells, demonstrating the importance of both sites for mRNA decay.
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39
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Clayton CE. Networks of gene expression regulation in Trypanosoma brucei. Mol Biochem Parasitol 2014; 195:96-106. [PMID: 24995711 DOI: 10.1016/j.molbiopara.2014.06.005] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Revised: 06/19/2014] [Accepted: 06/23/2014] [Indexed: 10/25/2022]
Abstract
Regulation of gene expression in Kinetoplastids relies mainly on post-transcriptional mechanisms. Recent high-throughput analyses, combined with mathematical modelling, have demonstrated possibilities for transcript-specific regulation at every stage: trans splicing, polyadenylation, translation, and degradation of both the precursor and the mature mRNA. Different mRNA degradation pathways result in different types of degradation kinetics. The original idea that the fate of an mRNA - or even just its degradation kinetics - can be defined by a single "regulatory element" is an over-simplification. It is now clear that every mRNA can bind many different proteins, some of which may compete with each other. Superimposed upon this complexity are the interactions of those proteins with effectors of gene expression. The amount of protein that is made from a gene is therefore determined by a complex network of interactions.
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Affiliation(s)
- C E Clayton
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
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40
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Young DJ, Stoddart A, Nakitandwe J, Chen SC, Qian Z, Downing JR, Le Beau MM. Knockdown of Hnrnpa0, a del(5q) gene, alters myeloid cell fate in murine cells through regulation of AU-rich transcripts. Haematologica 2014; 99:1032-40. [PMID: 24532040 DOI: 10.3324/haematol.2013.098657] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The control of mRNA stability plays a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation and tumorigenesis. HNRNPA0, which encodes an RNA-binding protein shown to regulate transcript stability via binding to the AU-rich elements of mRNAs, is located within the commonly deleted segment of 5q31.2 in myeloid neoplasms with a del(5q), and is expressed at haploinsufficient levels in these patients. We show that HNRNPA0 is normally highly expressed in hematopoietic stem cells and exhibits dynamic changes in expression during the course of differentiation. To model HNRNPA0 haploinsufficiency, we used RNAi interference in primary murine cells and an experimental cell system, and found that reduced Hnrnpa0 expression leads to a shift from monocytic towards granulocytic differentiation. Microarray-based global expression profiling revealed that Hnrnpa0 knockdown disproportionally impacts AU-rich containing transcripts and alters expression of myeloid specification genes. In therapy-related myeloid neoplasms with a del(5q), AU-rich containing mRNAs are enriched in transcripts that encode proteins associated with increased growth and proliferation. Our findings implicate haploinsufficiency of HNRNPA0 as one of the key initiating mutations in the pathogenesis of myeloid neoplasms with a del(5q), and suggest that therapies that target AU-rich elements warrant consideration in efforts to develop new mechanism-based treatment strategies.
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Affiliation(s)
- David J Young
- Department of Pediatrics, Division of Oncology, Johns Hopkins University, Baltimora, MD
| | - Angela Stoddart
- Department of Medicine and the Comprehensive Cancer Center, University of Chicago, IL
| | - Joy Nakitandwe
- St. Jude Children's Research Hospital, Memphis, Tennessee
| | | | - Zhijian Qian
- University of Illinois Cancer Center, Chicago, IL, USA
| | | | - Michelle M Le Beau
- Department of Medicine and the Comprehensive Cancer Center, University of Chicago, IL
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41
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Mahmoud L, Al-Enezi F, Al-Saif M, Warsy A, Khabar KSA, Hitti EG. Sustained stabilization of Interleukin-8 mRNA in human macrophages. RNA Biol 2014; 11:124-33. [PMID: 24525793 DOI: 10.4161/rna.27863] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The mRNAs of most inflammatory mediators are short-lived due to AU-rich elements (AREs) in their 3'-untranslated regions. AREs ensure a low basal level of expression during homeostasis and a transient nature of expression during the inflammatory response. Here, we report that the mRNA of the pro-inflammatory chemokine IL-8, which contains an archetypal ARE, is unexpectedly constitutively abundant and highly stable in primary human monocytes and macrophages. Using the pre-monocyte-like THP-1 cell line that can differentiate into macrophage-like cells, we show that a low level of unstable IL-8 mRNA in undifferentiated cells (half-life<30 min) becomes constitutively elevated and the mRNA is dramatically stabilized in differentiated THP-1 cells with a half-life of more than 15 h similar to primary monocytes and macrophages. In contrast, the level and stability of TNF-α mRNA also containing an ARE is only slightly affected by differentiation; it remains low and unstable in primary macrophages and differentiated THP-1 cells with an estimated half-life of less than 20 min. This differentiation-dependent stabilization of IL-8 mRNA is p38 MAPK-independent and is probably coupled with reduced protein translation. Reporter assays in THP-1 cells suggest that the ARE alone is not sufficient for the constitutive stabilization in macrophage-like cells and imply an effect of the natural biogenesis of the transcript on the stabilization of the mature form. We present a novel, cell type-dependent sustained stabilization of an ARE-containing mRNA with similarities to situations found in disease.
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Affiliation(s)
- Linah Mahmoud
- Molecular Biomedicine Program; King Faisal Specialist Hospital and Research Center; Riyadh, Saudi Arabia
| | - Fatma Al-Enezi
- Molecular Biomedicine Program; King Faisal Specialist Hospital and Research Center; Riyadh, Saudi Arabia; Department of Biochemistry; King Saud University; Riyadh, Saudi Arabia
| | - Maher Al-Saif
- Molecular Biomedicine Program; King Faisal Specialist Hospital and Research Center; Riyadh, Saudi Arabia
| | - Arjumand Warsy
- Department of Biochemistry; King Saud University; Riyadh, Saudi Arabia
| | - Khalid S A Khabar
- Molecular Biomedicine Program; King Faisal Specialist Hospital and Research Center; Riyadh, Saudi Arabia
| | - Edward G Hitti
- Molecular Biomedicine Program; King Faisal Specialist Hospital and Research Center; Riyadh, Saudi Arabia
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42
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Liu MT, Nagre NN, Ryan K. Structurally diverse low molecular weight activators of the mammalian pre-mRNA 3' cleavage reaction. Bioorg Med Chem 2014; 22:834-41. [PMID: 24373842 PMCID: PMC4018835 DOI: 10.1016/j.bmc.2013.12.006] [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: 09/20/2013] [Revised: 11/23/2013] [Accepted: 12/03/2013] [Indexed: 11/15/2022]
Abstract
The 3' end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the downstream boundary of the 3' untranslated region, which in many genes carries regulatory sequences. A large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase negatively regulates the 3' cleavage reaction, we have found a variety of new small molecule activators of the in vitro reconstituted pre-mRNA 3' cleavage reaction. New activators include a cyclic peptide PPM1D inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved l-arginine β-naphthylamide analog. The minimal concentration required for in vitro cleavage has been improved from 200μM to the 200nM-100μM range. These compounds provide unexpected leads in the search for small molecule tools able to affect pre-mRNA 3' end formation.
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Affiliation(s)
- Min Ting Liu
- Department of Chemistry, The City College of New York, The City University of New York, New York, NY 10031, USA
| | - Nagaraja N Nagre
- Department of Chemistry, The City College of New York, The City University of New York, New York, NY 10031, USA
| | - Kevin Ryan
- Department of Chemistry, The City College of New York, The City University of New York, New York, NY 10031, USA.
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43
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Emerging roles for ribonucleoprotein modification and remodeling in controlling RNA fate. Trends Cell Biol 2013; 23:504-10. [PMID: 23756094 DOI: 10.1016/j.tcb.2013.05.001] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2013] [Revised: 05/01/2013] [Accepted: 05/02/2013] [Indexed: 12/26/2022]
Abstract
In the cell, mRNAs and non-coding RNAs exist in association with proteins to form ribonucleoprotein (RNP) complexes. Regulation of RNP stability and function is achieved by alterations to the RNP through poorly understood mechanisms into which recent studies have now begun to provide insight. This emerging body of work points to chemical modification of RNPs at the RNA or protein level and ATP-dependent RNP remodeling by RNA helicases/RNA-dependent ATPases as central events that dictate RNA fate. Some RNP modifications serve as tags for recruitment of regulatory proteins, with RNP modifiers and recruited proteins analogous to the writers and readers of chromatin modification, respectively. This review highlights examples in which RNP modification and ATP-dependent remodeling play key roles in the control of eukaryotic RNA fate, suggesting that we are only at the beginning of uncovering the multitude of ways in which RNP modification and remodeling impact RNA regulation.
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44
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Brooks SA, Blackshear PJ. Tristetraprolin (TTP): interactions with mRNA and proteins, and current thoughts on mechanisms of action. BIOCHIMICA ET BIOPHYSICA ACTA 2013; 1829:666-79. [PMID: 23428348 PMCID: PMC3752887 DOI: 10.1016/j.bbagrm.2013.02.003] [Citation(s) in RCA: 303] [Impact Index Per Article: 25.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/24/2012] [Revised: 01/25/2013] [Accepted: 02/04/2013] [Indexed: 12/14/2022]
Abstract
Changes in mRNA stability and translation are critical control points in the regulation of gene expression, particularly genes encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenosine and uridine (AU)-rich elements (ARE), often located in the 3' untranslated regions (3'UTR) of mRNAs, are known to target transcripts for rapid decay. They are also involved in the regulation of mRNA stability and translation in response to extracellular cues. This review focuses on one of the best characterized ARE binding proteins, tristetraprolin (TTP), the founding member of a small family of CCCH tandem zinc finger proteins. In this survey, we have reviewed the current status of TTP interactions with mRNA and proteins, and discussed current thinking about TTP's mechanism of action to promote mRNA decay. We also review the proposed regulation of TTP's functions by phosphorylation. Finally, we have discussed emerging evidence for TTP operating as a translational regulator. This article is part of a Special Issue entitled: RNA Decay mechanisms.
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Affiliation(s)
- Seth A. Brooks
- Veterans Affairs Medical Center, White River Junction, Vermont, USA
- Department of Medicine, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire, USA
| | - Perry J. Blackshear
- The Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
- Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina USA
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45
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Briata P, Chen CY, Ramos A, Gherzi R. Functional and molecular insights into KSRP function in mRNA decay. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2012. [PMID: 23178464 DOI: 10.1016/j.bbagrm.2012.11.003] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
KSRP is a single strand nucleic acid binding protein that controls gene expression at multiple levels. In this review we focus on the recent molecular, cellular, and structural insights into the mRNA decay promoting function of KSRP. We discuss also some aspects of KSRP-dependent microRNA maturation from precursors that are related to its mRNA destabilizing function. This article is part of a Special Issue entitled: RNA Decay mechanisms.
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Affiliation(s)
- Paola Briata
- IRCCS Azienda Ospedaliera Universitaria San Martino, Genova, Italy
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46
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Chan CS, Ming-Lum A, Golds GB, Lee SJ, Anderson RJ, Mui ALF. Interleukin-10 inhibits lipopolysaccharide-induced tumor necrosis factor-α translation through a SHIP1-dependent pathway. J Biol Chem 2012; 287:38020-7. [PMID: 22955274 DOI: 10.1074/jbc.m112.348599] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Production of the proinflammatory cytokine TNFα by activated macrophages is an important component of host defense. However, TNFα production must be tightly controlled to avoid pathological consequences. The anti-inflammatory cytokine IL-10 inhibits TNFα mRNA expression through activation of the STAT3 transcription factor pathway and subsequent expression of STAT3-dependent gene products. We hypothesized that IL-10 must also have more rapid mechanisms of action and show that IL-10 rapidly shifts existing TNFα mRNA from polyribosome-associated polysomes to monosomes. This translation suppression requires the presence of SHIP1 (SH2 domain-containing inositol 5'-phosphatase 1) and involves inhibition of Mnk1 (MAPK signal-integrating kinase 1). Furthermore, activating SHIP1 using a small-molecule agonist mimics the inhibitory effect of IL-10 on Mnk1 phosphorylation and TNFα translation. Our data support the existence of an alternative STAT3-independent pathway through SHIP1 for IL-10 to regulate TNFα translation during the anti-inflammatory response.
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Affiliation(s)
- Catherine S Chan
- Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, Vancouver, British Columbia, Canada
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47
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Cancer cell growth suppression by a 62nt AU-rich RNA from C/EBPβ 3'UTR through competitive binding with HuR. Biochem Biophys Res Commun 2012; 426:122-8. [PMID: 22921787 DOI: 10.1016/j.bbrc.2012.08.049] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2012] [Accepted: 08/10/2012] [Indexed: 01/29/2023]
Abstract
AU-rich elements are functional motifs in the 3'untranslated region of mRNA and are binding sites for the RNA binding protein HuR, an mRNA stabilizer and translation enhancer implicated in carcinogenesis. It is not clear whether, and, if so, how the AU-rich elements function in cells when they are separated from their mRNA and form an independent RNA species. Here, we show that a short RNA with AU-rich elements derived from C/EBPβ 3'UTR suppressed growth in a human liver cancer cell line. It specifically bound HuR, and it competed with C/EBPβ mRNA in order to bind to HuR. Our results provide evidence that the cancer cell growth suppression by this 62nt RNA containing AU-rich elements may be due to competitive binding to HuR. This work may open new options for the development of novel anti-cancer drugs.
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