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Chou YS, Logeswaran D, Chow CN, L Dunn P, Podlevsky JD, Liu T, Akhter K, Chen JJL. A degenerate telomerase RNA directs telomeric DNA synthesis in lepidopteran insects. Proc Natl Acad Sci U S A 2025; 122:e2424443122. [PMID: 40020192 DOI: 10.1073/pnas.2424443122] [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: 11/22/2024] [Accepted: 01/29/2025] [Indexed: 03/08/2025] Open
Abstract
Telomerase elongates telomeres to maintain chromosome stability in most eukaryotes. Despite extensive studies across eukaryotic kingdoms, the telomerase holoenzyme in arthropods remains poorly understood. In this study, we purify the telomerase ribonucleoprotein complex from the lepidopteran insect Spodoptera frugiperda (fall armyworm) and identify a copurified 135-nucleotide telomerase RNA (TR) component. This miniature S. frugiperda TR (sfTR), the smallest TR known to date, retains a universal pseudoknot structure and a structurally defined template. Despite its small size, sfTR assembles with the recombinant S. frugiperda telomerase reverse transcriptase (sfTERT) protein in vivo to reconstitute telomerase activity for the synthesis of insect telomeric DNA repeats (TTAGG)n. The sfTR gene, like other animal TR genes, features an snRNA-type RNA polymerase II promoter. Uniquely, the sfTR transcript harbors a 5'-7-methylguanosine (M7G) cap, as opposed to the more typical snRNA-type 2,2,7-trimethylguanosine (TMG) cap. The difference in 5'-cap is likely because sfTR lacks the H/ACA snoRNA biogenesis domain necessary for cap hypermethylation. Moreover, sfTR also lacks the CR4/5 regulatory domain that is indispensable in vertebrate TRs for telomerase activity. This degenerate sfTR complements an enigmatic sfTERT that is missing certain telomerase-specific elements yet catalytically active in the absence of sfTR. Thus, insects have evolved a simplified telomerase, consisting of a small noncoding RNA that retains only minimal attributes essential for telomerase function. The simplified insect telomerase demonstrates a plausible evolutionary pathway for the emergence of telomerase ribonucleoprotein complex, arising from an ancient reverse transcriptase associated with a simple templating RNA component in early eukaryotes.
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Affiliation(s)
- Yu-Shu Chou
- School of Molecular Sciences, Arizona State University, Tempe, AZ 85281
| | | | - Chi-Nga Chow
- School of Molecular Sciences, Arizona State University, Tempe, AZ 85281
| | - Phoebe L Dunn
- School of Life Sciences, Arizona State University, Tempe, AZ 85281
| | | | - Tianxiang Liu
- School of Molecular Sciences, Arizona State University, Tempe, AZ 85281
| | - Khadiza Akhter
- School of Molecular Sciences, Arizona State University, Tempe, AZ 85281
| | - Julian J-L Chen
- School of Molecular Sciences, Arizona State University, Tempe, AZ 85281
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2
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Shou S, Maolan A, Zhang D, Jiang X, Liu F, Li Y, Zhang X, Geer E, Pu Z, Hua B, Guo Q, Zhang X, Pang B. Telomeres, telomerase, and cancer: mechanisms, biomarkers, and therapeutics. Exp Hematol Oncol 2025; 14:8. [PMID: 39871386 PMCID: PMC11771031 DOI: 10.1186/s40164-025-00597-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2025] [Accepted: 01/15/2025] [Indexed: 01/29/2025] Open
Abstract
Telomeres and telomerase play crucial roles in the initiation and progression of cancer. As biomarkers, they aid in distinguishing benign from malignant tissues. Despite the promising therapeutic potential of targeting telomeres and telomerase for therapy, translating this concept from the laboratory to the clinic remains challenging. Many candidate drugs remain in the experimental stage, with only a few advancing to clinical trials. This review explores the relationship between telomeres, telomerase, and cancer, synthesizing their roles as biomarkers and reviewing the outcomes of completed trials. We propose that changes in telomere length and telomerase activity can be used to stratify cancer stages. Furthermore, we suggest that differential expression of telomere and telomerase components at the subcellular level holds promise as a biomarker. From a therapeutic standpoint, combining telomerase-targeted therapies with drugs that mitigate the adverse effects of telomerase inhibition may offer a viable strategy.
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Affiliation(s)
- Songting Shou
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Ayidana Maolan
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Di Zhang
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Xiaochen Jiang
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Fudong Liu
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Yi Li
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Xiyuan Zhang
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - En Geer
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Zhenqing Pu
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Baojin Hua
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
| | - Qiujun Guo
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
| | - Xing Zhang
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
| | - Bo Pang
- Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
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3
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Arata Y, Jurica P, Parrish N, Sako Y. Bioinformatic Annotation of Transposon DNA Processing Genes on the Long-Read Genome Assembly of Caenorhabditis elegans. Bioinform Biol Insights 2024; 18:11779322241304668. [PMID: 39713040 PMCID: PMC11662393 DOI: 10.1177/11779322241304668] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2024] [Accepted: 11/13/2024] [Indexed: 12/24/2024] Open
Abstract
Transposable elements (TEs) or transposons are thought to play roles in animal physiological processes, such as germline, early embryonic, and brain development, as well as aging. However, their roles have not been systematically investigated through experimental studies. In this study, we created a catalog of genes directly involved in replication, excision, or integration of transposon-coding DNA, which we refer to as transposon DNA processing genes (TDPGs). Specifically, to bridge the gap to experimental studies, we sought potentially functional TDPGs which maintain intact open reading frames and the amino acids at their catalytic cores on the latest long-read genome assembly of Caenorhabditis elegans, VC2010. Among 52 519 TE loci, we identified 145 potentially functional TDPGs encoded in long terminal repeat elements, long interspersed nuclear elements, terminal inverted repeat elements, Helitrons, and Mavericks/Polintons. Our TDPG catalog, which contains a feasible number of genes, allows for the experimental manipulation of TE mobility in vivo, regardless of whether the TEs are autonomous or non-autonomous, thereby potentially promoting the study of the physiological functions of TE mobility.
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Affiliation(s)
- Yukinobu Arata
- Cellular Informatics Laboratory, Cluster for Pioneering Research (CPR), RIKEN, Saitama, Japan
| | - Peter Jurica
- Cellular Informatics Laboratory, Cluster for Pioneering Research (CPR), RIKEN, Saitama, Japan
| | - Nicholas Parrish
- Genome Immunobiology RIKEN Hakubi Research Team, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
| | - Yasushi Sako
- Cellular Informatics Laboratory, Cluster for Pioneering Research (CPR), RIKEN, Saitama, Japan
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4
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Kageler L, Aquilanti E. Discovery of telomerase inhibitors: existing strategies and emerging innovations. Biochem Soc Trans 2024; 52:1957-1968. [PMID: 39194999 DOI: 10.1042/bst20230264] [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: 02/08/2024] [Revised: 08/05/2024] [Accepted: 08/06/2024] [Indexed: 08/29/2024]
Abstract
Telomerase, crucial for maintaining telomere length, is an attractive target for cancer therapy due to its role in cellular immortality. Despite three decades of research efforts, no small-molecule telomerase inhibitors have been clinically approved, highlighting the extensive challenges in developing effective telomerase-based therapeutics. This review examines conventional and emerging methods to measure telomerase activity and discusses existing inhibitors, including oligonucleotides and small molecules. Furthermore, this review highlights recent breakthroughs in structural studies of telomerase using cryo-electron microscopy, which can facilitate improved structure-based drug design. Altogether, advancements in structural methodologies and high-throughput screening offer promising prospects for telomerase-based cancer therapeutic development.
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Affiliation(s)
- Lauren Kageler
- Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, U.S.A
| | - Elisa Aquilanti
- Division of Neuro Oncology, Dana Farber Cancer Institute, Boston, MA, U.S.A
- Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, U.S.A
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA, U.S.A
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5
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Carabias A, Camara-Wilpert S, Mestre MR, Lopéz-Méndez B, Hendriks IA, Zhao R, Pape T, Fuglsang A, Luk SHC, Nielsen ML, Pinilla-Redondo R, Montoya G. Retron-Eco1 assembles NAD +-hydrolyzing filaments that provide immunity against bacteriophages. Mol Cell 2024; 84:2185-2202.e12. [PMID: 38788717 DOI: 10.1016/j.molcel.2024.05.001] [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] [Received: 09/26/2023] [Revised: 03/15/2024] [Accepted: 05/01/2024] [Indexed: 05/26/2024]
Abstract
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
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Affiliation(s)
- Arturo Carabias
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
| | - Sarah Camara-Wilpert
- Section of Microbiology, Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark
| | - Mario Rodríguez Mestre
- Section of Microbiology, Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark
| | - Blanca Lopéz-Méndez
- Protein Purification and Characterization Facility, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Ivo A Hendriks
- Proteomics Department, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Ruiliang Zhao
- Section of Microbiology, Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark
| | - Tillmann Pape
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark; Core Facility for Integrated Microscopy (CFIM), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Anders Fuglsang
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Sean Hoi-Ching Luk
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Michael L Nielsen
- Proteomics Department, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Rafael Pinilla-Redondo
- Section of Microbiology, Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark.
| | - Guillermo Montoya
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
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6
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Liu M, Zhang Y, Jian Y, Gu L, Zhang D, Zhou H, Wang Y, Xu ZX. The regulations of telomerase reverse transcriptase (TERT) in cancer. Cell Death Dis 2024; 15:90. [PMID: 38278800 PMCID: PMC10817947 DOI: 10.1038/s41419-024-06454-7] [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: 09/22/2023] [Revised: 01/04/2024] [Accepted: 01/08/2024] [Indexed: 01/28/2024]
Abstract
Abnormal activation of telomerase occurs in most cancer types, which facilitates escaping from cell senescence. As the key component of telomerase, telomerase reverse transcriptase (TERT) is regulated by various regulation pathways. TERT gene changing in its promoter and phosphorylation respectively leads to TERT ectopic expression at the transcription and protein levels. The co-interacting factors play an important role in the regulation of TERT in different cancer types. In this review, we focus on the regulators of TERT and these downstream functions in cancer regulation. Determining the specific regulatory mechanism will help to facilitate the development of a cancer treatment strategy that targets telomerase and cancer cell senescence. As the most important catalytic subunit component of telomerase, TERT is rapidly regulated by transcriptional factors and PTM-related activation. These changes directly influence TERT-related telomere maintenance by regulating telomerase activity in telomerase-positive cancer cells, telomerase assembly with telomere-binding proteins, and recruiting telomerase to the telomere. Besides, there are also non-canonical functions that are influenced by TERT, including the basic biological functions of cancer cells, such as proliferation, apoptosis, cell cycle regulation, initiating cell formation, EMT, and cell invasion. Other downstream effects are the results of the influence of transcriptional factors by TERT. Currently, some small molecular inhibitors of TERT and TERT vaccine are under research as a clinical therapeutic target. Purposeful work is in progress.
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Affiliation(s)
- Mingdi Liu
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun, 130021, Jilin, China
| | - Yuning Zhang
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun, 130021, Jilin, China
| | - Yongping Jian
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun, 130021, Jilin, China
| | - Liting Gu
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun, 130021, Jilin, China
| | - Dan Zhang
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun, 130021, Jilin, China
| | - Honglan Zhou
- Department of Urology, The First Hospital of Jilin University, Changchun, 130021, Jilin, China.
| | - Yishu Wang
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun, 130021, Jilin, China.
| | - Zhi-Xiang Xu
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun, 130021, Jilin, China.
- Department of Urology, The First Hospital of Jilin University, Changchun, 130021, Jilin, China.
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7
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Oh GS, An S, Kim S. Harnessing CRISPR-Cas adaptation for RNA recording and beyond. BMB Rep 2024; 57:40-49. [PMID: 38053290 PMCID: PMC10828431] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 04/04/2023] [Accepted: 04/04/2023] [Indexed: 12/07/2023] Open
Abstract
Prokaryotes encode clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) genes as an adaptive immune machinery. CRISPR-Cas systems effectively protect hosts from the invasion of foreign enemies, such as bacteriophages and plasmids. During a process called 'adaptation', non-self-nucleic acid fragments are acquired as spacers between repeats in the host CRISPR array, to establish immunological memory. The highly conserved Cas1-Cas2 complexes function as molecular recorders to integrate spacers in a time course manner, which can subsequently be expressed as crRNAs complexed with Cas effector proteins for the RNAguided interference pathways. In some of the RNA-targeting type III systems, Cas1 proteins are fused with reverse transcriptase (RT), indicating that RT-Cas1-Cas2 complexes can acquire RNA transcripts for spacer acquisition. In this review, we summarize current studies that focus on the molecular structure and function of the RT-fused Cas1-Cas2 integrase, and its potential applications as a directional RNA-recording tool in cells. Furthermore, we highlight outstanding questions for RT-Cas1-Cas2 studies and future directions for RNA-recording CRISPR technologies. [BMB Reports 2024; 57(1): 40-49].
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Affiliation(s)
- Gyeong-Seok Oh
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
| | - Seongjin An
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
- Department of Life Sciences, School of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea
| | - Sungchul Kim
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
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8
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Oh GS, An S, Kim S. Harnessing CRISPR-Cas adaptation for RNA recording and beyond. BMB Rep 2024; 57:40-49. [PMID: 38053290 PMCID: PMC10828431 DOI: 10.5483/bmbrep.2023-0050] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 04/04/2023] [Accepted: 04/04/2023] [Indexed: 03/09/2025] Open
Abstract
Prokaryotes encode clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) genes as an adaptive immune machinery. CRISPR-Cas systems effectively protect hosts from the invasion of foreign enemies, such as bacteriophages and plasmids. During a process called 'adaptation', non-self-nucleic acid fragments are acquired as spacers between repeats in the host CRISPR array, to establish immunological memory. The highly conserved Cas1-Cas2 complexes function as molecular recorders to integrate spacers in a time course manner, which can subsequently be expressed as crRNAs complexed with Cas effector proteins for the RNAguided interference pathways. In some of the RNA-targeting type III systems, Cas1 proteins are fused with reverse transcriptase (RT), indicating that RT-Cas1-Cas2 complexes can acquire RNA transcripts for spacer acquisition. In this review, we summarize current studies that focus on the molecular structure and function of the RT-fused Cas1-Cas2 integrase, and its potential applications as a directional RNA-recording tool in cells. Furthermore, we highlight outstanding questions for RT-Cas1-Cas2 studies and future directions for RNA-recording CRISPR technologies. [BMB Reports 2024; 57(1): 40-49].
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Affiliation(s)
- Gyeong-Seok Oh
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
| | - Seongjin An
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
- Department of Life Sciences, School of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea
| | - Sungchul Kim
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
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9
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Özmaldar A, Balta B. Formation and Effects of Upstream DNA-RNA Base Pairing in Telomerase. Chembiochem 2023; 24:e202300501. [PMID: 37743538 DOI: 10.1002/cbic.202300501] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Revised: 09/04/2023] [Accepted: 09/22/2023] [Indexed: 09/26/2023]
Abstract
Telomere elongation by telomerase consists of two types of translocation: duplex translocation during each repeat synthesis and template translocation at the end of repeat synthesis. Our replica exchange molecular dynamics simulations show that in addition to the Watson-Crick interactions in the active site, templating RNA can also form base pairs with the upstream regions of DNA, mostly with the second upstream DNA repeat with respect to the 3'-end. At the end of the repeat synthesis, dG10-P442 and dG11-N446 hydrogen bonds form. Then, active-site base pairs dissociate one by one, and the RNA bases reanneal with the complementary base on the upstream DNA repeat. For each dissociating base pair a new one forms, thus conserving the number of base pairs during template translocation.
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Affiliation(s)
- Aydın Özmaldar
- Department of Molecular Biology and Genetics, Istanbul Technical University, Maslak, 34469, Istanbul, Turkey
| | - Bülent Balta
- Department of Molecular Biology and Genetics, Istanbul Technical University, Maslak, 34469, Istanbul, Turkey
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10
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da Silva VL, de Paiva SC, de Oliveira HC, Fernandes CAH, Salvador GHM, Fontes MRDM, Cano MIN. Biochemical and structural characterization of the RT domain of Leishmania sp. telomerase reverse transcriptase. Biochim Biophys Acta Gen Subj 2023; 1867:130451. [PMID: 37751810 DOI: 10.1016/j.bbagen.2023.130451] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Revised: 07/22/2023] [Accepted: 08/25/2023] [Indexed: 09/28/2023]
Abstract
BACKGROUND The Leishmania genus comprises parasites that cause leishmaniasis, a neglected disease spread worldwide. Leishmania sp. telomeres are composed of TTAGGG repeats maintained by telomerase. In most eukaryotes, the enzyme minimal complex contains the TER (telomerase RNA) and the TERT (telomerase reverse transcriptase) components. The TERT holds the enzyme catalytic core and is formed by four structural and functional domains (TEN, Telomerase Essential N-terminal; TRBD, Telomerase RNA Binding Domain; RT, the reverse transcriptase domain and CTE, C-Terminal Extension domain). METHODS AND RESULTS Amino acid sequence alignments, protein structure prediction analysis, and protein: nucleic acid interaction assays were used to show that the Leishmania major RT domain preserves the canonical structural elements found in higher eukaryotes, including the canonical motifs and the aspartic acid residues that stabilize the Mg2+ ion cofactor. Furthermore, amino acid substitutions specific to the Leishmania genus and partial conservation of the residues involved with nucleic acid interactions are shown. The purified recombinant Leishmania RT protein is biochemically active and interacts with the G-rich telomeric strand and the TER template sequence. CONCLUSION Our results highlight that the telomerase catalysis mechanism is conserved in a pathogen of medical importance despite the structural peculiarities present in the parasite's RT domain.
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Affiliation(s)
- Vitor Luiz da Silva
- Department of Chemical and Biological Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil
| | - Stephany Cacete de Paiva
- Department of Chemical and Biological Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil
| | - Hamine Cristina de Oliveira
- Department of Biophysics and Pharmacology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil
| | - Carlos Alexandre H Fernandes
- UMR 7590, CNRS, Muséum National d'Histoire Naturelle, IRD, Institut de Minéralogie, Physique des Matériaux et de Cosmochimie, IMPMC, Sorbonne Université, Paris, France
| | | | - Marcos Roberto de M Fontes
- Department of Biophysics and Pharmacology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil; Institute for Advanced Studies of the Sea (IEAMAR), São Paulo State University (UNESP), São Vicente, SP, Brazil
| | - Maria Isabel N Cano
- Department of Chemical and Biological Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil.
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11
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Das CK, Gupta A, Nair NN. Probing the general base for DNA polymerization in telomerase: a molecular dynamics investigation. Phys Chem Chem Phys 2023; 25:14147-14157. [PMID: 37162325 DOI: 10.1039/d3cp00521f] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/11/2023]
Abstract
Telomerase is an RNA-dependent DNA polymerase that plays a role in the maintenance of the 3' end of the eukaryotic chromosome, known as a telomere, by catalyzing the DNA polymerization reaction in cancer and embryonic stem cells. The detailed molecular details of the DNA polymerization by telomerase, especially the general base for deprotonating the terminal 3'-hydroxyl, which triggers the chemical reaction, remain elusive. We conducted a computational investigation using hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations to probe the detailed mechanism of the reaction. Our simulations started with the telomerase:RNA:DNA:dNTP ternary complex, and by using enhanced sampling QM/MM MD simulations, we probed the general base involved directly in the polymerization. We report the participation of an aspartate (Asp344) coordinated to Mg and an active site water molecule, jointly acting as a base during nucleic acid addition. The Asp344 residue remains transiently protonated during the course of the reaction, and later it deprotonates by transferring its proton to the water at the end of the reaction.
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Affiliation(s)
- Chandan Kumar Das
- Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, 208016, India.
| | - Abhinav Gupta
- Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, 208016, India.
| | - Nisanth N Nair
- Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, 208016, India.
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12
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Huber LB, Betz K, Marx A. Reverse Transcriptases: From Discovery and Applications to Xenobiology. Chembiochem 2023; 24:e202200521. [PMID: 36354312 DOI: 10.1002/cbic.202200521] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2022] [Revised: 11/09/2022] [Indexed: 11/12/2022]
Abstract
Reverse transcriptases are DNA polymerases that can use RNA as a template for DNA synthesis. They thus catalyze the reverse of transcription. Although discovered in 1970, reverse transcriptases are still of great interest and are constantly being further developed for numerous modern research approaches. They are frequently used in biotechnological and molecular diagnostic applications. In this review, we describe the discovery of these fascinating enzymes and summarize research results and applications ranging from molecular cloning, direct virus detection, and modern sequencing methods to xenobiology.
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Affiliation(s)
- Luisa B Huber
- Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78464, Konstanz, Germany
| | - Karin Betz
- Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78464, Konstanz, Germany
| | - Andreas Marx
- Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78464, Konstanz, Germany
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13
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Karami Fath M, Garousi S, Mottahedi M, Ghasemzadeh N, Salmani K, Olfati F, Beit Saeed M, Sotoudeh S, Barati G. The role of hypoxia-inducible factors in breast cancer stem cell specification. Pathol Res Pract 2023; 243:154349. [PMID: 36791562 DOI: 10.1016/j.prp.2023.154349] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/24/2022] [Revised: 01/24/2023] [Accepted: 01/27/2023] [Indexed: 01/31/2023]
Abstract
Breast tumor is heterogeneous cancer with high morbidity and mortality rates, particularly in developing countries. Despite new efforts to reduce the breast cancer implications, the number of newly diagnosed cases is increasing worldwide. It is believed that cancer stem cells (CSCs) are responsible for the implication of cancers including breast cancer. Although CSCs compose a small population in tumor bulks, they play a crucial role in tumor initiation, progression, metastasis, and chemotherapeutic resistance. These events are mediated by the hypoxia-inducible factor (HIF) pathway which regulates the transcription of genes involved in CSC maintenance and tumorigenesis. In this review, we discussed the mechanisms by which hypoxia- or chemotherapy-induced HIFs promote breast CSC specification.
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Affiliation(s)
- Mohsen Karami Fath
- Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
| | - Setareh Garousi
- Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mehran Mottahedi
- Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | | | - Kiana Salmani
- Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
| | - Fatemeh Olfati
- Department of Reproductive Health, Faculty of Nursing and Midwifery, Tehran University of Medical Sciences, Tehran, Iran
| | - Miad Beit Saeed
- Faculty of Nursing and Midwifery, Abadan Islamic Azad University, Abadan, Iran
| | - Sina Sotoudeh
- Faculty of Nursing and Midwifery, Guilan University of Medical Sciences, Guilan, Iran
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14
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Liu Y, Betori RC, Pagacz J, Frost GB, Efimova EV, Wu D, Wolfgeher DJ, Bryan TM, Cohen SB, Scheidt KA, Kron SJ. Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization. Cell Chem Biol 2022; 29:1517-1531.e7. [PMID: 36206753 PMCID: PMC9588800 DOI: 10.1016/j.chembiol.2022.09.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 07/29/2022] [Accepted: 09/15/2022] [Indexed: 11/03/2022]
Abstract
Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT's non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT's role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.
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Affiliation(s)
- Yue Liu
- Ludwig Center for Metastasis Research and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
| | - Rick C Betori
- Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
| | - Joanna Pagacz
- Ludwig Center for Metastasis Research and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
| | - Grant B Frost
- Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
| | - Elena V Efimova
- Ludwig Center for Metastasis Research and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
| | - Ding Wu
- Ludwig Center for Metastasis Research and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
| | - Donald J Wolfgeher
- Ludwig Center for Metastasis Research and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
| | - Tracy M Bryan
- Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia
| | - Scott B Cohen
- Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia
| | - Karl A Scheidt
- Department of Chemistry, Northwestern University, Evanston, IL 60208, USA.
| | - Stephen J Kron
- Ludwig Center for Metastasis Research and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
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15
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Wang Q, Feng J, Tang L. Non-Coding RNA Related to MAPK Signaling Pathway in Liver Cancer. Int J Mol Sci 2022; 23:11908. [PMID: 36233210 PMCID: PMC9570382 DOI: 10.3390/ijms231911908] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2022] [Revised: 10/03/2022] [Accepted: 10/04/2022] [Indexed: 11/16/2022] Open
Abstract
The advancement in high-throughput sequencing analysis and the evaluation of chromatin state maps have revealed that eukaryotic cells produce many non-coding transcripts/RNAs. Further, a strong association was observed between some non-coding RNAs and cancer development. The mitogen-activated protein kinases (MAPK) belong to the serine-threonine kinase family and are the primary signaling pathways involved in cell proliferation from the cell surface to the nucleus. They play an important role in various human diseases. A few non-coding RNAs associated with the MAPK signaling pathway play a significant role in the development of several malignancies, including liver cancer. In this review, we summarize the molecular mechanisms and interactions of microRNA, lncRNA, and other non-coding RNAs in the development of liver cancer that are associated with the MAPK signaling pathway. Further, we briefly discuss the therapeutic strategies for liver cancer related to ncRNA and the MAPK signaling pathway.
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Affiliation(s)
- Qiuxia Wang
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China
| | - Jianguo Feng
- Department of Anesthesiology, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
- Laboratory of Anesthesiology, Southwest Medical University, Luzhou 646000, China
| | - Liling Tang
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China
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16
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Functional Interactions of Kluyveromyces lactis Telomerase Reverse Transcriptase with the Three-Way Junction and the Template Domains of Telomerase RNA. Int J Mol Sci 2022; 23:ijms231810757. [PMID: 36142669 PMCID: PMC9504884 DOI: 10.3390/ijms231810757] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 09/08/2022] [Accepted: 09/13/2022] [Indexed: 11/23/2022] Open
Abstract
The ribonucleoprotein telomerase contains two essential components: telomerase RNA (TER) and telomerase reverse transcriptase (TERT, Est2 in yeast). A small portion of TER, termed the template, is copied by TERT onto the chromosome ends, thus compensating for sequence loss due to incomplete DNA replication and nuclease action. Although telomerase RNA is highly divergent in sequence and length across fungi and mammals, structural motifs essential for telomerase function are conserved. Here, we show that Est2 from the budding yeast Kluyveromyces lactis (klEst2) binds specifically to an essential three-way junction (TWJ) structure in K. lactis TER, which shares a conserved structure and sequence features with the essential CR4-CR5 domain of vertebrate telomerase RNA. klEst2 also binds specifically to the template domain, independently and mutually exclusive of its interaction with TWJ. Furthermore, we present the high-resolution structure of the klEst2 telomerase RNA-binding domain (klTRBD). Mutations introduced in vivo in klTRBD based on the solved structure or in TWJ based on its predicted RNA structure caused severe telomere shortening. These results demonstrate the conservation and importance of these domains and the multiple protein–RNA interactions between Est2 and TER for telomerase function.
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17
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Jácome R, Campillo-Balderas JA, Becerra A, Lazcano A. Structural Analysis of Monomeric RNA-Dependent Polymerases Revisited. J Mol Evol 2022; 90:283-295. [PMID: 35639164 PMCID: PMC9153872 DOI: 10.1007/s00239-022-10059-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2022] [Accepted: 05/11/2022] [Indexed: 12/24/2022]
Abstract
In the past few years, our understanding of the RNA virosphere has changed dramatically due to the growth and spurt of metagenomics, exponentially increasing the number of RNA viral sequences, and providing a better understanding of their range of potential hosts. As of today, the only conserved protein among RNA viruses appears to be the monomeric RNA-dependent RNA polymerase. This enzyme belongs to the right-hand DNA-and RNA polymerases, which also includes reverse transcriptases and eukaryotic replicative DNA polymerases. The ubiquity of this protein in RNA viruses makes it a unique evolutionary marker and an appealing broad-spectrum antiviral target. In this work pairwise structural comparisons of viral RdRps and RTs were performed, including tertiary structures that have been obtained in the last few years. The resulting phylogenetic tree shows that the RdRps from (+)ss- and dsRNA viruses might have been recruited several times throughout the evolution of mobile genetic elements. RTs also display multiple evolutionary routes. We have identified a structural core comprising the entire palm, a large moiety of the fingers and the N-terminal helices of the thumb domain, comprising over 300 conserved residues, including two regions that we have named the “knuckles” and the “hypothenar eminence”. The conservation of an helix bundle in the region preceding the polymerase domain confirms that (−)ss and dsRNA Reoviruses’ polymerases share a recent ancestor. Finally, the inclusion of DNA polymerases into our structural analyses suggests that monomeric RNA-dependent polymerases might have diverged from B-family polymerases.
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Affiliation(s)
- Rodrigo Jácome
- Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico, Mexico
| | | | - Arturo Becerra
- Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico, Mexico
| | - Antonio Lazcano
- Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico, Mexico.
- Miembro de El Colegio Nacional, Mexico, Mexico.
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18
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Pimentel SC, Upton HE, Collins K. Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase. J Biol Chem 2022; 298:101624. [PMID: 35065960 PMCID: PMC8857657 DOI: 10.1016/j.jbc.2022.101624] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Revised: 01/12/2022] [Accepted: 01/13/2022] [Indexed: 11/16/2022] Open
Abstract
Broad evolutionary expansion of polymerase families has enabled specialization of their activities for distinct cellular roles. In addition to template-complementary synthesis, many polymerases extend their duplex products by nontemplated nucleotide addition (NTA). This activity is exploited for laboratory strategies of cloning and sequencing nucleic acids and could have important biological function, although the latter has been challenging to test without separation-of-function mutations. Several retroelement and retroviral reverse transcriptases (RTs) support NTA and also template jumping, by which the RT performs continuous complementary DNA (cDNA) synthesis using physically separate templates. Previous studies that aimed to dissect the relationship between NTA and template jumping leave open questions about structural requirements for each activity and their interdependence. Here, we characterize the structural requirements for cDNA synthesis, NTA, template jumping, and the unique terminal transferase activity of Bombyx mori R2 non-long terminal repeat retroelement RT. With sequence alignments and structure modeling to guide mutagenesis, we generated enzyme variants across motifs generally conserved or specific to RT subgroups. Enzyme variants had diverse NTA profiles not correlated with other changes in cDNA synthesis activity or template jumping. Using these enzyme variants and panels of activity assay conditions, we show that template jumping requires NTA. However, template jumping by NTA-deficient enzymes can be rescued using primer duplex with a specific length of 3′ overhang. Our findings clarify the relationship between NTA and template jumping as well as additional activities of non-long terminal repeat RTs, with implications for the specialization of RT biological functions and laboratory applications.
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Affiliation(s)
- Sydney C Pimentel
- Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA
| | - Heather E Upton
- Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA
| | - Kathleen Collins
- Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA.
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19
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Flexibility of telomerase in binding the RNA template and DNA telomeric repeat. Proc Natl Acad Sci U S A 2022; 119:2116159118. [PMID: 34969861 PMCID: PMC8740718 DOI: 10.1073/pnas.2116159118] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/22/2021] [Indexed: 12/12/2022] Open
Abstract
Telomerase reverse transcriptase (TERT) has a conserved central cavity near the active site. Using enzymatic and mutagenesis analyses, we provide experimental evidence that an artificially looped-out telomeric repeat in the DNA primer can be transiently accommodated in the cavity of Tribolium castaneum (tcTERT). Kinetically, tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. An RNA duplex downstream of the RNA-template region after a flexible linker enhances the efficiency of primer extension by tcTERT. In addition to the peripheral cavities that accommodate looped-out RNA during each telomeric repeat synthesis, the central cavity that can accommodate the looped-out DNA may aid RNA-template translocation between cycles of telomeric repeat synthesis. Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3′ end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5′ template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA–DNA binding likely underlies the processivity of telomeric repeat addition.
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20
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Davis JA, Chakrabarti K. Telomerase ribonucleoprotein and genome integrity-An emerging connection in protozoan parasites. WILEY INTERDISCIPLINARY REVIEWS. RNA 2021; 13:e1710. [PMID: 34973045 DOI: 10.1002/wrna.1710] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Revised: 12/08/2021] [Accepted: 12/10/2021] [Indexed: 12/20/2022]
Abstract
Telomerase has an established role in telomere maintenance in eukaryotes. However, recent studies have begun to implicate telomerase in cellular roles beyond telomere maintenance. Specifically, evidence is emerging of cross-talks between telomerase mediated telomere homeostasis and DNA repair pathways. Telomere shortening due to the end replication problem is a constant threat to genome integrity in eukaryotic cells. This poses a particular problem in unicellular parasitic protists because their major virulence genes are located at the subtelomeric loci. Although telomerase is the major regulator of telomere lengthening in eukaryotes, it is less studied in the ancient eukaryotes, including clinically important human pathogens. Recent research is highlighting interplay between telomerase and the DNA damage response in human parasites. The importance of this interplay in pathogen virulence is only beginning to be illuminated, including the potential to highlight novel developmental regulation of telomerase in parasites who transition between multiple developmental stages throughout their life cycle. In this review, we will discuss the telomerase ribonucleoprotein enzyme and DNA repair pathways with emerging views in human parasites to give a broader perspective of the possible connection of telomere, telomerase, and DNA repair pathways across eukaryotic lineages and highlight their potential role in pathogen virulence. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
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Affiliation(s)
| | - Kausik Chakrabarti
- University of North Carolina at Charlotte, Charlotte, North Carolina, USA
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21
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Nguyen THD. Structural biology of human telomerase: progress and prospects. Biochem Soc Trans 2021; 49:1927-1939. [PMID: 34623385 PMCID: PMC8589416 DOI: 10.1042/bst20200042] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 09/08/2021] [Accepted: 09/10/2021] [Indexed: 12/28/2022]
Abstract
Telomerase ribonucleoprotein was discovered over three decades ago as a specialized reverse transcriptase that adds telomeric repeats to the ends of linear eukaryotic chromosomes. Telomerase plays key roles in maintaining genome stability; and its dysfunction and misregulation have been linked to different types of cancers and a spectrum of human genetic disorders. Over the years, a wealth of genetic and biochemical studies of human telomerase have illuminated its numerous fascinating features. Yet, structural studies of human telomerase have lagged behind due to various challenges. Recent technical developments in cryo-electron microscopy have allowed for the first detailed visualization of the human telomerase holoenzyme, revealing unprecedented insights into its active site and assembly. This review summarizes the cumulative work leading to the recent structural advances, as well as highlights how the future structural work will further advance our understanding of this enzyme.
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Affiliation(s)
- Thi Hoang Duong Nguyen
- Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K
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22
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Russi M, Marson D, Fermeglia A, Aulic S, Fermeglia M, Laurini E, Pricl S. The fellowship of the RING: BRCA1, its partner BARD1 and their liaison in DNA repair and cancer. Pharmacol Ther 2021; 232:108009. [PMID: 34619284 DOI: 10.1016/j.pharmthera.2021.108009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2021] [Revised: 08/22/2021] [Accepted: 09/20/2021] [Indexed: 12/12/2022]
Abstract
The breast cancer type 1 susceptibility protein (BRCA1) and its partner - the BRCA1-associated RING domain protein 1 (BARD1) - are key players in a plethora of fundamental biological functions including, among others, DNA repair, replication fork protection, cell cycle progression, telomere maintenance, chromatin remodeling, apoptosis and tumor suppression. However, mutations in their encoding genes transform them into dangerous threats, and substantially increase the risk of developing cancer and other malignancies during the lifetime of the affected individuals. Understanding how BRCA1 and BARD1 perform their biological activities therefore not only provides a powerful mean to prevent such fatal occurrences but can also pave the way to the development of new targeted therapeutics. Thus, through this review work we aim at presenting the major efforts focused on the functional characterization and structural insights of BRCA1 and BARD1, per se and in combination with all their principal mediators and regulators, and on the multifaceted roles these proteins play in the maintenance of human genome integrity.
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Affiliation(s)
- Maria Russi
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Domenico Marson
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Alice Fermeglia
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Suzana Aulic
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Maurizio Fermeglia
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Erik Laurini
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Sabrina Pricl
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy; Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland.
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23
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HIF-1 recruits NANOG as a coactivator for TERT gene transcription in hypoxic breast cancer stem cells. Cell Rep 2021; 36:109757. [PMID: 34592152 DOI: 10.1016/j.celrep.2021.109757] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2020] [Revised: 07/07/2021] [Accepted: 09/01/2021] [Indexed: 12/12/2022] Open
Abstract
Breast cancer stem cells (BCSCs) play essential roles in tumor formation, drug resistance, relapse, and metastasis. NANOG is a protein required for stem cell self-renewal, but the mechanisms by which it performs this function are poorly understood. Here, we show that hypoxia-inducible factor 1α (HIF-1α) is required for NANOG-mediated BCSC enrichment. Mechanistically, NANOG is recruited by HIF-1 to cooperatively activate transcription of the TERT gene encoding the telomerase reverse transcriptase that maintains telomere length, which is required for stem cell self-renewal. NANOG stimulates HIF-1 transcriptional activity by recruitment of the deubiquitinase USP9X, which inhibits HIF-1α protein degradation, and by stabilizing HIF-1α interaction with the coactivator p300, which mediates histone acetylation. Our results delineate a cooperative transcriptional mechanism by which HIF-1 and NANOG mediate BCSC self-renewal.
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24
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Xie X, Li M, Zhou M, Chow SF, Tsang CK. Pharmacological preconditioning by TERT inhibitor BIBR1532 confers neuronal ischemic tolerance through TERT-mediated transcriptional reprogramming. J Neurochem 2021; 159:690-709. [PMID: 34532857 DOI: 10.1111/jnc.15515] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Revised: 08/25/2021] [Accepted: 09/13/2021] [Indexed: 11/30/2022]
Abstract
After a sublethal ischemic preconditioning (IPC) stimulus, the brain has a remarkable capability of acquiring tolerance to subsequent ischemic insult by establishing precautionary self-protective mechanism. Understanding this endogenous mechanism would reveal novel and effective neuroprotective targets for ischemic brain injury. Our previous study has implied that telomerase reverse transcriptase (TERT) is associated with IPC-induced tolerance. Here, we investigated the mechanism of TERT-mediated ischemic tolerance. Preconditioning was modeled by oxygen-glucose deprivation (OGD) and by TERT inhibitor BIBR1532 in primary neurons. We found that ischemic tolerance was conferred by BIBR1532 preconditioning. We used the Cleavage-Under-Targets-And-Tagmentation approach, a recently developed method with superior signal-to-noise ratio, to comprehensively map the genomic binding sites of TERT in primary neurons, and showed that more than 50% of TERT-binding sites were located at the promoter regions. Mechanistically, we demonstrated that under normal conditions TERT physically bound to many previously unknown genomic loci in neurons, whereas BIBR1532 preconditioning significantly altered TERT-chromatin-binding profile. Intriguingly, we found that BIBR1532-preconditioned neurons showed significant up-regulation of promoter binding of TERT to the mitochondrial anti-oxidant genes, which were correlated with their elevated expression. Functional analysis further indicated that BIBR1532-preconditioning significantly reduced ROS levels and enhanced tolerance to severe ischemia-induced mitochondrial oxidative stress in neurons in a TERT-dependent manner. Together, these results demonstrate that BIBR1532 confers neuronal ischemic tolerance through TERT-mediated transcriptional reprogramming for up-regulation of mitochondrial anti-oxidation gene expression, suggesting the translational potential of BIBR1532 as a therapeutic agent for the treatment of cerebral ischemic injury and oxidative stress-induced neurological disorders.
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Affiliation(s)
- Xuemin Xie
- Clinical Neuroscience Institute, The First Affiliated Hospital of Jinan University, Guangzhou, China.,Department of Neurology, The First Clinical Medical School of Jinan University, Guangzhou, China
| | - Mingxi Li
- Clinical Neuroscience Institute, The First Affiliated Hospital of Jinan University, Guangzhou, China.,Department of Neurology, The First Clinical Medical School of Jinan University, Guangzhou, China
| | - Mengyao Zhou
- Clinical Neuroscience Institute, The First Affiliated Hospital of Jinan University, Guangzhou, China.,Core Research Center, The First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Shing Fung Chow
- Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong Special Administrative Region, Hong Kong, China
| | - Chi Kwan Tsang
- Clinical Neuroscience Institute, The First Affiliated Hospital of Jinan University, Guangzhou, China
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25
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Plyasova AA, Berrino E, Khan II, Veselovsky AV, Pokrovsky VS, Angeli A, Ferraroni M, Supuran CT, Pokrovskaya MV, Alexandrova SS, Gladilina YA, Sokolov NN, Hilal A, Carta F, Zhdanov DD. Mechanisms of the Antiproliferative and Antitumor Activity of Novel Telomerase-Carbonic Anhydrase Dual-Hybrid Inhibitors. J Med Chem 2021; 64:11432-11444. [PMID: 34283610 DOI: 10.1021/acs.jmedchem.1c00756] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Human (h) telomerase (TL; EC 2.7.7.49) plays a key role in sustaining cancer cells by means of elongating telomeric repeats at the 3' ends of chromosomes. Since TL-inhibitor (TI) stand-alone cancer therapy has been proven to be remarkably challenging, a polypharmacological approach represents a valid alternative. Here we consider a series of compounds able to inhibit both hTL and the tumor-associated carbonic anhydrases (CAs; EC 4.2.1.1) IX and XII. Compounds 7 and 9 suppressed hTL activity in both cell lysates and human colon cancer cell lines, and prolonged incubation with either 7 or 9 resulted in telomere shortening, cell cycle arrest, replicative senescence, and apoptosis. Enzyme kinetics showed that 7 and 9 are mixed-type inhibitors of the binding of DNA primers and deoxynucleoside triphosphate (dNTP) to the TL catalytic subunit hTERT, which is in agreement with docking experiments. Compound 9 showed antitumor activity in Colo-205 mouse xenografts and suppressed telomerase activity by telomere reduction.
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Affiliation(s)
- Anna A Plyasova
- Institute of Biomedical Chemistry, Pogodinskaya Street 10/8, Moscow 119121, Russia
| | - Emanuela Berrino
- Dipartimento di Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Florence, Italy
| | - Irina I Khan
- Peoples' Friendship University of Russia (RUDN University), Miklukho-Maklaya Street 6, Moscow 117198, Russia.,N.N. Blokhin Cancer Research Center, Kashirskoe Shosse 24, Moscow 115478, Russia
| | | | - Vadim S Pokrovsky
- Peoples' Friendship University of Russia (RUDN University), Miklukho-Maklaya Street 6, Moscow 117198, Russia.,N.N. Blokhin Cancer Research Center, Kashirskoe Shosse 24, Moscow 115478, Russia
| | - Andrea Angeli
- Dipartimento di Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Florence, Italy
| | - Marta Ferraroni
- Dipartimento di Chimica "Ugo Schiff", Università degli Studi di Firenze, Via della Lastruccia 3-13, 50019 Florence, Italy
| | - Claudiu T Supuran
- Dipartimento di Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Florence, Italy
| | - Marina V Pokrovskaya
- Institute of Biomedical Chemistry, Pogodinskaya Street 10/8, Moscow 119121, Russia
| | | | - Yulia A Gladilina
- Institute of Biomedical Chemistry, Pogodinskaya Street 10/8, Moscow 119121, Russia
| | - Nikolay N Sokolov
- Institute of Biomedical Chemistry, Pogodinskaya Street 10/8, Moscow 119121, Russia
| | - Abdullah Hilal
- Institute of Biomedical Chemistry, Pogodinskaya Street 10/8, Moscow 119121, Russia
| | - Fabrizio Carta
- Dipartimento di Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Florence, Italy
| | - Dmitry D Zhdanov
- Institute of Biomedical Chemistry, Pogodinskaya Street 10/8, Moscow 119121, Russia
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26
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Zhai LT, Rety S, Chen WF, Song ZY, Auguin D, Sun B, Dou SX, Xi XG. Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡. Nucleic Acids Res 2021; 49:4768-4781. [PMID: 33856462 PMCID: PMC8096264 DOI: 10.1093/nar/gkab261] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 03/28/2021] [Accepted: 04/05/2021] [Indexed: 12/12/2022] Open
Abstract
Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.
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Affiliation(s)
- Liu-Tao Zhai
- State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Stephane Rety
- University Lyon, ENS de Lyon, University Claude Bernard, CNRS UMR 5239, INSERM U1210, LBMC, 46 Allée d’Italie Site Jacques Monod, F-69007 Lyon, France
| | - Wei-Fei Chen
- State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Ze-Yu Song
- State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Daniel Auguin
- Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), Université d’Orléans, INRA, USC1328, 45067 Orléans; Structural Motility, Institut Curie, CNRS, UMR 144 Paris, France
| | - Bo Sun
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Shuo-Xing Dou
- Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
- School of Physical Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xu-Guang Xi
- State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
- Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), UMR 8113 CNRS, Institut D’Alembert, École Normale Supérieure Paris-Saclay, Université Paris-Saclay, 4, Avenue des Sciences, 91190 Gif sur Yvette, France
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27
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Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex. Nat Commun 2021; 12:2571. [PMID: 33958590 PMCID: PMC8102632 DOI: 10.1038/s41467-021-22900-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 03/26/2021] [Indexed: 02/03/2023] Open
Abstract
CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.
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28
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Gala K, Khattar E. Long non-coding RNAs at work on telomeres: Functions and implications in cancer therapy. Cancer Lett 2021; 502:120-132. [PMID: 33450357 DOI: 10.1016/j.canlet.2020.12.036] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2020] [Revised: 12/13/2020] [Accepted: 12/29/2020] [Indexed: 12/27/2022]
Abstract
Long non-coding RNAs (lncRNAs) are known to regulate various biological processes including cancer. Cancer cells possess limitless replicative potential which is attained by telomere length maintenance while normal somatic cells have a limited lifespan because their telomeres shorten with every cell division ultimately triggering replicative senescence. Two lncRNAs have been observed to play a key role in telomere length maintenance. First is the lncRNA TERC (telomerase RNA component) which functions as a template for telomeric DNA synthesis in association with telomerase reverse transcriptase (TERT) which serves as the catalytic component. Together they constitute the telomerase complex which functions as a reverse transcriptase to elongate telomeres. Second lncRNA that helps in regulating telomere length is the telomeric repeat-containing RNA (TERRA) which is transcribed from the subtelomeric region and extends to the telomeric region. TERC and TERRA exhibit important functions in cancer with implications in precision oncology. In this review, we discuss various aspects of these important lncRNAs in humans and their role in cancer along with recent advancements in their anticancer therapeutic application.
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Affiliation(s)
- Kavita Gala
- Sunandan Divatia School of Science, SVKM's NMIMS (Deemed to be) University, Mumbai, 400056, Maharashtra, India
| | - Ekta Khattar
- Sunandan Divatia School of Science, SVKM's NMIMS (Deemed to be) University, Mumbai, 400056, Maharashtra, India.
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29
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Cheng HP, Deumens E, Freericks JK, Li C, Sanders BA. Application of Quantum Computing to Biochemical Systems: A Look to the Future. Front Chem 2020; 8:587143. [PMID: 33330375 PMCID: PMC7732423 DOI: 10.3389/fchem.2020.587143] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2020] [Accepted: 10/15/2020] [Indexed: 12/24/2022] Open
Abstract
Chemistry is considered as one of the more promising applications to science of near-term quantum computing. Recent work in transitioning classical algorithms to a quantum computer has led to great strides in improving quantum algorithms and illustrating their quantum advantage. Because of the limitations of near-term quantum computers, the most effective strategies split the work over classical and quantum computers. There is a proven set of methods in computational chemistry and materials physics that has used this same idea of splitting a complex physical system into parts that are treated at different levels of theory to obtain solutions for the complete physical system for which a brute force solution with a single method is not feasible. These methods are variously known as embedding, multi-scale, and fragment techniques and methods. We review these methods and then propose the embedding approach as a method for describing complex biochemical systems, with the parts not only treated with different levels of theory, but computed with hybrid classical and quantum algorithms. Such strategies are critical if one wants to expand the focus to biochemical molecules that contain active regions that cannot be properly explained with traditional algorithms on classical computers. While we do not solve this problem here, we provide an overview of where the field is going to enable such problems to be tackled in the future.
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Affiliation(s)
- Hai-Ping Cheng
- Quantum Theory Project, Department of Physics, University of Florida, Gainesville, FL, United States
| | - Erik Deumens
- Quantum Theory Project, Department of Physics, University of Florida, Gainesville, FL, United States
| | - James K. Freericks
- Department of Physics, Georgetown University, Washington, DC, United States
| | - Chenglong Li
- Department of Medicinal Chemistry, University of Florida, Gainesville, FL, United States
| | - Beverly A. Sanders
- Department of Computer and Information Science and Engineering, University of Florida, Gainesville, FL, United States
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30
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A structurally conserved human and Tetrahymena telomerase catalytic core. Proc Natl Acad Sci U S A 2020; 117:31078-31087. [PMID: 33229538 DOI: 10.1073/pnas.2011684117] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Telomerase is a ribonucleoprotein complex that counteracts the shortening of chromosome ends due to incomplete replication. Telomerase contains a catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). However, what defines TERT and separates it from other reverse transcriptases remains a subject of debate. A recent cryoelectron microscopy map of Tetrahymena telomerase revealed the structure of a previously uncharacterized TERT domain (TRAP) with unanticipated interactions with the telomerase essential N-terminal (TEN) domain and roles in telomerase activity. Both TEN and TRAP are absent in the putative Tribolium TERT that has been used as a model for telomerase for over a decade. To investigate the conservation of TRAP and TEN across species, we performed multiple sequence alignments and statistical coupling analysis on all identified TERTs and find that TEN and TRAP have coevolved as telomerase-specific domains. Integrating the data from bioinformatic analysis and the structure of Tetrahymena telomerase, we built a pseudoatomic model of human telomerase catalytic core that accounts for almost all of the cryoelectron microscopy density in a published map, including TRAP in previously unassigned density as well as telomerase RNA domains essential for activity. This more complete model of the human telomerase catalytic core illustrates how domains of TER and TERT, including the TEN-TRAP complex, can interact in a conserved manner to regulate telomere synthesis.
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31
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Abstract
We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.
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32
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Kim SS, Baek GO, Ahn HR, Sung S, Seo CW, Cho HJ, Nam SW, Cheong JY, Eun JW. Serum small extracellular vesicle-derived LINC00853 as a novel diagnostic marker for early hepatocellular carcinoma. Mol Oncol 2020; 14:2646-2659. [PMID: 32525601 PMCID: PMC7530776 DOI: 10.1002/1878-0261.12745] [Citation(s) in RCA: 54] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2020] [Revised: 05/25/2020] [Accepted: 06/04/2020] [Indexed: 02/06/2023] Open
Abstract
This study aimed to identify novel long noncoding RNA (lncRNA) biomarkers for hepatocellular carcinoma (HCC) using publicly available tissue genomic datasets and validate their diagnostic utility for early-stage HCC. Differentially expressed lncRNAs between 371 HCC and 50 nontumor tissues were obtained from The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC) project. Subsequently, the expression of the serum- and extracellular vesicle (EV)-derived lncRNA was assessed in 10 patients with HCC and 10 healthy controls using RT-qPCR. The candidate lncRNAs were validated in 90 HCC and 92 non-HCC (29 healthy control, 28 chronic hepatitis, 35 liver cirrhosis) patients. The sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) were calculated for the candidate lncRNAs and the current HCC biomarker, alpha-fetoprotein (AFP). SFTA1P, HOTTIP, HAGLROS, LINC01419, HAGLR, CRNDE, and LINC00853 were markedly upregulated in HCC in TCGA_LIHC dataset. Among them, LINC00853 has not been reported in relation to HCC before. In patients with HCC, only expression of small EV-derived LINC00853 (EV-LINC00853) was increased. EV-LINC00853 showed excellent discriminatory ability in the diagnosis of all-stage HCC (AUC = 0.934, 95% confidence interval = 0.887-0.966). Moreover, using a 14-fold increase and 20 ng·mL-1 as cutoffs for EV-LINC00853 expression and AFP level, respectively, EV-LINC00853 was found to have a sensitivity of 93.75% and specificity of 89.77%, while AFP showed only 9.38% sensitivity and 72.73% specificity for the diagnosis of early-stage HCC (mUICC stage I). EV-LINC00853 had a positivity of 97% and 67% in AFP-negative and AFP-positive early HCC, respectively. Serum EV-derived LINC00853 may be a novel potential diagnostic biomarker for early HCC, especially for AFP-negative HCC.
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Affiliation(s)
- Soon Sun Kim
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea
| | - Geum Ok Baek
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea
| | - Hye Ri Ahn
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Sciences, Ajou University Graduate School of Medicine, Suwon, South Korea
| | - Suna Sung
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Sciences, Ajou University Graduate School of Medicine, Suwon, South Korea
| | - Chul Won Seo
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Sciences, Ajou University Graduate School of Medicine, Suwon, South Korea
| | - Hyo Jung Cho
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea
| | - Suk Woo Nam
- Functional RNomics Research Center, The Catholic University of Korea, Seoul, South Korea.,Department of Biomedicine & Health Sciences, Graduate School of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Jae Youn Cheong
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea
| | - Jung Woo Eun
- Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea
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33
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Gandhi M, Groß M, Holler JM, Coggins SA, Patil N, Leupold JH, Munschauer M, Schenone M, Hartigan CR, Allgayer H, Kim B, Diederichs S. The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer. Nat Commun 2020; 11:3214. [PMID: 32587247 PMCID: PMC7316977 DOI: 10.1038/s41467-020-17007-9] [Citation(s) in RCA: 81] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2019] [Accepted: 06/05/2020] [Indexed: 02/06/2023] Open
Abstract
Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.
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Affiliation(s)
- Minakshi Gandhi
- Division of RNA Biology & Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
- German Academic Exchange Service (DAAD), Bonn, Germany; Helmholtz International Graduate School for Cancer Research (HIGS), Heidelberg, Germany; Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
| | - Matthias Groß
- Division of RNA Biology & Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Jessica M Holler
- Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA, USA
| | - Si'Ana A Coggins
- Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA, USA
| | - Nitin Patil
- Department of Experimental Surgery-Cancer Metastasis, Medical Faculty Mannheim, Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Mannheim, Germany
| | - Joerg H Leupold
- Department of Experimental Surgery-Cancer Metastasis, Medical Faculty Mannheim, Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Mannheim, Germany
| | | | | | | | - Heike Allgayer
- Department of Experimental Surgery-Cancer Metastasis, Medical Faculty Mannheim, Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Mannheim, Germany
| | - Baek Kim
- Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA, USA
- Center for Drug Discovery, Children's Healthcare of Atlanta, Atlanta, GA, USA
| | - Sven Diederichs
- Division of RNA Biology & Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.
- Division of Cancer Research, Department of Thoracic Surgery, Medical Center, Faculty of Medicine, University of Freiburg, German Cancer Consortium (DKTK)-Partner Site Freiburg, Freiburg, Germany.
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34
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Schaich MA, Sanford SL, Welfer GA, Johnson SA, Khoang TH, Opresko PL, Freudenthal BD. Mechanisms of nucleotide selection by telomerase. eLife 2020; 9:55438. [PMID: 32501800 PMCID: PMC7274783 DOI: 10.7554/elife.55438] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Accepted: 05/18/2020] [Indexed: 01/31/2023] Open
Abstract
Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.
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Affiliation(s)
- Matthew A Schaich
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, United States
| | - Samantha L Sanford
- Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, and UPMC Hillman Cancer Center, Pittsburgh, United States
| | - Griffin A Welfer
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, United States
| | - Samuel A Johnson
- Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, and UPMC Hillman Cancer Center, Pittsburgh, United States
| | - Thu H Khoang
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, United States
| | - Patricia L Opresko
- Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, and UPMC Hillman Cancer Center, Pittsburgh, United States
| | - Bret D Freudenthal
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, United States.,Department of Cancer Biology, University of Kansas Medical Center, Kansas City, United States
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35
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Jonchhe S, Pandey S, Karna D, Pokhrel P, Cui Y, Mishra S, Sugiyama H, Endo M, Mao H. Duplex DNA Is Weakened in Nanoconfinement. J Am Chem Soc 2020; 142:10042-10049. [PMID: 32383870 PMCID: PMC7295077 DOI: 10.1021/jacs.0c01978] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
For proteins and DNA secondary structures such as G-quadruplexes and i-motifs, nanoconfinement can facilitate their folding and increase structural stabilities. However, the properties of the physiologically prevalent B-DNA duplex have not been elucidated inside the nanocavity. Using a 17-bp DNA duplex in the form of a hairpin stem, here, we probed folding and unfolding transitions of the hairpin DNA duplex inside a DNA origami nanocavity. Compared to the free solution, the DNA hairpin inside the nanocage with a 15 × 15 nm cross section showed a drastic decrease in mechanical (20 → 9 pN) and thermodynamic (25 → 6 kcal/mol) stabilities. Free energy profiles revealed that the activation energy of unzipping the hairpin DNA duplex decreased dramatically (28 → 8 kcal/mol), whereas the transition state moved closer to the unfolded state inside the nanocage. All of these indicate that nanoconfinement weakens the stability of the hairpin DNA duplex to an unexpected extent. In a DNA hairpin made of a stem that contains complementary telomeric G-quadruplex (GQ) and i-motif (iM) forming sequences, formation of the Hoogsteen base pairs underlining the GQ or iM is preferred over the Watson-Crick base pairs in the DNA hairpin. These results shed light on the behavior of DNA in nanochannels, nanopores, or nanopockets of various natural or synthetic machineries. It also elucidates an alternative pathway to populate noncanonical DNA over B-DNA in the cellular environment where the nanocavity is abundant.
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Affiliation(s)
- Sagun Jonchhe
- Department of Chemistry & Biochemistry, Kent State University, Kent, OH 44242, USA
| | - Shankar Pandey
- Department of Chemistry & Biochemistry, Kent State University, Kent, OH 44242, USA
| | - Deepak Karna
- Department of Chemistry & Biochemistry, Kent State University, Kent, OH 44242, USA
| | - Pravin Pokhrel
- Department of Chemistry & Biochemistry, Kent State University, Kent, OH 44242, USA
| | - Yunxi Cui
- Department of Chemistry & Biochemistry, Kent State University, Kent, OH 44242, USA
| | - Shubham Mishra
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan
- Institute for Integrated Cell–Material Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan
| | - Hiroshi Sugiyama
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan
- Institute for Integrated Cell–Material Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan
| | - Masayuki Endo
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan
- Institute for Integrated Cell–Material Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan
| | - Hanbin Mao
- Department of Chemistry & Biochemistry, Kent State University, Kent, OH 44242, USA
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36
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Baginski M, Serbakowska K. In silico design of telomerase inhibitors. Drug Discov Today 2020; 25:1213-1222. [PMID: 32387261 DOI: 10.1016/j.drudis.2020.04.024] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2019] [Revised: 04/23/2020] [Accepted: 04/29/2020] [Indexed: 12/31/2022]
Abstract
Telomerase is a reverse transcriptase enzyme involved in DNA synthesis at the end of linear chromosomes. Unlike in most other cells, telomerase is reactivated most cancerous cells and, therefore, has become a promising new anticancer target. Despite extensive research, direct telomerase inhibitors have yet not been introduced to the clinics because of the complexity of this enzyme. Structures of this protein from simple organisms and human homology models are currently available and have been used in structure-based drug design efforts to find potential inhibitors. Different is silico strategies have been applied and different chemical groups have been explored. Here, we provide an overview of recent discoveries.
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Affiliation(s)
- Maciej Baginski
- Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, 80-233 Gdansk, Poland.
| | - Katarzyna Serbakowska
- Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, 80-233 Gdansk, Poland
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37
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Cooperative Analysis of Structural Dynamics in RNA-Protein Complexes by Single-Molecule Förster Resonance Energy Transfer Spectroscopy. Molecules 2020; 25:molecules25092057. [PMID: 32354083 PMCID: PMC7248720 DOI: 10.3390/molecules25092057] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2020] [Revised: 03/31/2020] [Accepted: 04/13/2020] [Indexed: 12/24/2022] Open
Abstract
RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.
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38
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Betori RC, Liu Y, Mishra RK, Cohen SB, Kron SJ, Scheidt KA. Targeted Covalent Inhibition of Telomerase. ACS Chem Biol 2020; 15:706-717. [PMID: 32017522 DOI: 10.1021/acschembio.9b00945] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Telomerase is a ribonuceloprotein complex responsible for maintaining telomeres and protecting chromosomal integrity. The human telomerase reverse transcriptase (hTERT) is expressed in ∼90% of cancer cells where it confers the capacity for limitless proliferation. Along with its established role in telomere lengthening, telomerase also serves noncanonical extra-telomeric roles in oncogenic signaling, resistance to apoptosis, and enhanced DNA damage response. We report a new class of natural-product-inspired covalent inhibitors of telomerase that target the catalytic active site.
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Affiliation(s)
- Rick C. Betori
- Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States
| | - Yue Liu
- Ludwig Center for Metastasis Research, The University of Chicago, Chicago, Illinois 60637, United States
| | - Rama K. Mishra
- Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208, United States
| | - Scott B. Cohen
- Children’s Medical Research Institute, University of Sydney, Westmead, New South Wales 2145, Australia
| | - Stephen J. Kron
- Ludwig Center for Metastasis Research, The University of Chicago, Chicago, Illinois 60637, United States
| | - Karl A. Scheidt
- Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States
- Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, United States
- Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208, United States
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39
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Smith EM, Pendlebury DF, Nandakumar J. Structural biology of telomeres and telomerase. Cell Mol Life Sci 2020; 77:61-79. [PMID: 31728577 PMCID: PMC6986361 DOI: 10.1007/s00018-019-03369-x] [Citation(s) in RCA: 132] [Impact Index Per Article: 26.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2019] [Revised: 10/11/2019] [Accepted: 10/31/2019] [Indexed: 01/16/2023]
Abstract
Telomeres are protein-DNA complexes that protect chromosome ends from illicit ligation and resection. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA to counter telomere shortening. Human telomeres are composed of complexes between telomeric DNA and a six-protein complex known as shelterin. The shelterin proteins TRF1 and TRF2 provide the binding affinity and specificity for double-stranded telomeric DNA, while the POT1-TPP1 shelterin subcomplex coats the single-stranded telomeric G-rich overhang that is characteristic of all our chromosome ends. By capping chromosome ends, shelterin protects telomeric DNA from unwanted degradation and end-to-end fusion events. Structures of the human shelterin proteins reveal a network of constitutive and context-specific interactions. The shelterin protein-DNA structures reveal the basis for both the high affinity and DNA sequence specificity of these interactions, and explain how shelterin efficiently protects chromosome ends from genome instability. Several protein-protein interactions, many provided by the shelterin component TIN2, are critical for upholding the end-protection function of shelterin. A survey of these protein-protein interfaces within shelterin reveals a series of "domain-peptide" interactions that allow for efficient binding and adaptability towards new functions. While the modular nature of shelterin has facilitated its part-by-part structural characterization, the interdependence of subunits within telomerase has made its structural solution more challenging. However, the exploitation of several homologs in combination with recent advancements in cryo-EM capabilities has led to an exponential increase in our knowledge of the structural biology underlying telomerase function. Telomerase homologs from a wide range of eukaryotes show a typical retroviral reverse transcriptase-like protein core reinforced with elements that deliver telomerase-specific functions including recruitment to telomeres and high telomere-repeat addition processivity. In addition to providing the template for reverse transcription, the RNA component of telomerase provides a scaffold for the catalytic and accessory protein subunits, defines the limits of the telomeric repeat sequence, and plays a critical role in RNP assembly, stability, and trafficking. While a high-resolution definition of the human telomerase structure is only beginning to emerge, the quick pace of technical progress forecasts imminent breakthroughs in this area. Here, we review the structural biology surrounding telomeres and telomerase to provide a molecular description of mammalian chromosome end protection and end replication.
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Affiliation(s)
- Eric M Smith
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA
- Program in Chemical Biology, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Devon F Pendlebury
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA
- Program in Chemical Biology, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Jayakrishnan Nandakumar
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
- Program in Chemical Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
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40
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Real-Time Fluorescence-Based Approaches to Disentangle Mechanisms of a Protein's RNA Chaperone Activity. Methods Mol Biol 2020. [PMID: 31889252 DOI: 10.1007/978-1-0716-0231-7_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
RNA-binding proteins with an RNA chaperone activity exert either one or both of the following catalytic activities: (1) RNA annealing, i.e., the protein supports intra- as well as intermolecular RNA-RNA interactions and (2) strand displacement, i.e., the protein mediates the exchange of individual strands of a preexisting RNA duplex. To discriminate and further characterize these activities, it requires defined assay systems. These are based on entirely or partially complementary RNA oligonucleotides that are labeled with fluorescent and/or quencher dyes. The non-catalyzed and the protein-supported associations of the RNA molecules are followed by a real-time fluorescence resonance energy transfer (FRET) system. By site-specific modification of the RNAs or the protein, the substrate- and protein-specific parameters of the RNA chaperone activity can be explored and identified.In this chapter, we present strategies on the design of labeled RNA molecules to be used to characterize the activities of an RNA-binding protein and explain how to monitor progress curves of RNA annealing and strand displacement reactions in single cuvette or well-plate scales. We provide sets of equations and models to determine and analyze different types of reactions, e.g., by calculation of first- and second-order rate constants. Likewise, we demonstrate how to exploit these simple experimental setups to elucidate elementary principles of the reaction mechanisms performed by the protein of interest by applying basic kinetic applications, such as ARRHENIUS and linear free energy relationship analyses. These approaches will be explained by providing example plots and graphs from experiments investigating the RNA chaperone activities of the RNA-binding proteins NF90-NF45 and AUF1 p45.
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41
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Wang Y, Sušac L, Feigon J. Structural Biology of Telomerase. Cold Spring Harb Perspect Biol 2019; 11:cshperspect.a032383. [PMID: 31451513 DOI: 10.1101/cshperspect.a032383] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Telomerase is a DNA polymerase that extends the 3' ends of chromosomes by processively synthesizing multiple telomeric repeats. It is a unique ribonucleoprotein (RNP) containing a specialized telomerase reverse transcriptase (TERT) and telomerase RNA (TER) with its own template and other elements required with TERT for activity (catalytic core), as well as species-specific TER-binding proteins important for biogenesis and assembly (core RNP); other proteins bind telomerase transiently or constitutively to allow association of telomerase and other proteins with telomere ends for regulation of DNA synthesis. Here we describe how nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography of TER and protein domains helped define the structure and function of the core RNP, laying the groundwork for interpreting negative-stain and cryo electron microscopy (cryo-EM) density maps of Tetrahymena thermophila and human telomerase holoenzymes. As the resolution has improved from ∼30 Å to ∼5 Å, these studies have provided increasingly detailed information on telomerase architecture and mechanism.
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Affiliation(s)
- Yaqiang Wang
- Department of Chemistry and Biochemistry, University of California Los Angeles (UCLA), Los Angeles, California 90095-1569
| | - Lukas Sušac
- Department of Chemistry and Biochemistry, University of California Los Angeles (UCLA), Los Angeles, California 90095-1569
| | - Juli Feigon
- Department of Chemistry and Biochemistry, University of California Los Angeles (UCLA), Los Angeles, California 90095-1569
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42
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Díaz-Cartagena D, Hernández-Cancel G, Bracho-Rincón DP, González-Feliciano JA, Cunci L, González CI, Cabrera CR. Label-Free Telomerase Activity Detection via Electrochemical Impedance Spectroscopy. ACS OMEGA 2019; 4:16724-16732. [PMID: 31646217 PMCID: PMC6796945 DOI: 10.1021/acsomega.9b00783] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/21/2019] [Accepted: 05/16/2019] [Indexed: 05/14/2023]
Abstract
In the last decade, researchers have been searching for innovative platforms, methods, and techniques able to address recurring problems with the current cancer detection methods. Early disease detection, fast results, point-of-care sensing, and cost are among the most prevalent issues that need further exploration in this field. Herein, studies are focused on overcoming these problems by developing an electrochemical device able to detect telomerase as a cancer biomarker. Electrochemical platforms and techniques are more appealing for cancer detection, offering lower costs than the established cancer detection methods, high sensitivity inherent to the technique, rapid signal processing, and their capacity of being miniaturized. Therefore, Au interdigital electrodes and electrochemical impedance spectroscopy were used to detect telomerase activity in acute T cell leukemia. Different cancer cell concentrations were evaluated, and a detection limit of 1.9 × 105 cells/mL was obtained. X-ray photoelectron spectroscopy was used to characterize the telomerase substrate (TS) DNA probe self-assembled monolayer on gold electrode surfaces. Atomic force microscopy displayed three-dimensional images of the surface to establish a height difference of 9.0 nm between the bare electrode and TS-modified Au electrodes. The TS probe is rich in guanines, thus forming secondary structures known as G-quadruplex that can be triggered with a fluorescence probe. Confocal microscopy fluorescence images showed the formation of DNA G-quadruplex because of TS elongation by telomerase on the Au electrode surface. Moreover, electrodes exposed to telomerase containing 2',3'-dideoxyguanosine-5'-triphosphate (ddGTP) did not exhibit high fluorescence, as ddGTP is a telomerase inhibitor, thus making this device suitable for telomerase inhibitors capacity studies. The electrochemical method and Au microchip device may be developed as a biosensor for a point-of-care medical device.
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Affiliation(s)
- Diana
C. Díaz-Cartagena
- Department
of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico 00925-2537, United States
- Molecular
Sciences Research Center, University of
Puerto Rico, San Juan, Puerto Rico 00926, United States
| | - Griselle Hernández-Cancel
- Molecular
Sciences Research Center, University of
Puerto Rico, San Juan, Puerto Rico 00926, United States
| | - Dina P. Bracho-Rincón
- Molecular
Sciences Research Center, University of
Puerto Rico, San Juan, Puerto Rico 00926, United States
- Department
of Biology, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico 00931, United States
| | - José A. González-Feliciano
- Molecular
Sciences Research Center, University of
Puerto Rico, San Juan, Puerto Rico 00926, United States
| | - Lisandro Cunci
- School
of Natural Sciences and Technology, Universidad
Ana G. Méndez, Gurabo Campus, Gurabo, Puerto
Rico 00778, United
States
| | - Carlos I. González
- Molecular
Sciences Research Center, University of
Puerto Rico, San Juan, Puerto Rico 00926, United States
- Department
of Biology, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico 00931, United States
| | - Carlos R. Cabrera
- Department
of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico 00925-2537, United States
- Molecular
Sciences Research Center, University of
Puerto Rico, San Juan, Puerto Rico 00926, United States
- E-mail:
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43
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Jansson LI, Stone MD. Single-Molecule Analysis of Reverse Transcriptase Enzymes. Cold Spring Harb Perspect Biol 2019; 11:11/9/a032458. [PMID: 31481455 DOI: 10.1101/cshperspect.a032458] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The original discovery of enzymes that synthesize DNA using an RNA template appeared to contradict the central dogma of biology, in which information is transferred, in a unidirectional way, from DNA genes into RNA molecules. The paradigm-shifting discovery of RNA-dependent DNA polymerases, also called reverse transcriptases (RTs), reshaped existing views for how cells function; however, the scope of the impact RTs impose on biology had yet to be realized. In the decades of research since the early 1970s, the biomedical and biotechnological significance of retroviral RTs, as well as the evolutionarily related telomerase enzyme, has become exceedingly clear. One common theme that has emerged in the course of RT-related research is the central role of nucleic acid binding and dynamics during enzyme function. However, directly interrogating these dynamic properties is challenging because of the stochastic properties of biological macromolecules. In this review, we describe how the development of single-molecule biophysical techniques has opened new windows through which to observe the dynamic behavior of this remarkable class of enzymes. Specifically, we focus on how the powerful single-molecule Förster resonance energy transfer (FRET) method has been exploited to study the structure and function of the human immunodeficiency virus (HIV) RT and telomerase ribonucleoprotein (RNP) enzymes. These exciting studies have refined our understanding of RT catalysis, have revealed unforeseen structural rearrangements between RTs and their nucleic acid substrates, and have helped to characterize the mode of action of RT-inhibiting drugs. We conclude with a discussion of how the ongoing development of single-molecule technologies will continue to empower researchers to probe RT mechanisms in new and exciting ways.
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Affiliation(s)
- Linnea I Jansson
- Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064.,The Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064
| | - Michael D Stone
- Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064.,The Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064
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44
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Yuan T, Zhao W, Niu Y, Fu Y, Lu L, Niu D. Exploration of the temporal-spatial expression pattern and DNA methylation-related regulation of the duck telomerase reverse transcriptase gene. Poult Sci 2019; 98:3257-3267. [PMID: 31064004 DOI: 10.3382/ps/pez240] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2018] [Accepted: 04/09/2019] [Indexed: 12/14/2022] Open
Abstract
Telomerase reverse transcriptase (TERT) is a catalytic subunit of telomerase that adds TTAGGG repeats to the 3'-overhang of telomeres. In the present study, we detected that the duck TERT (dTERT) gene was highly expressed in small intestine and kidney, followed by heart, leg muscle, spleen, pancreas, gonad, and liver at neonatal stage. From embryonic to neonatal stage, the highest dTERT mRNA in liver appeared at stage E19 (19 days at embryonic stage), while for the leg muscle the maximum expression occurred at E26. We also measured the relative telomerase activity (RTA) and relative telomere length (RTL) in the examined tissues and found that the changed tendency of RTA and RTL was not very consistent with that of TERT. In silico analysis revealed that there were three CpG islands (S1, S2, and S3) within the 5' regulatory region of the dTERT gene. Bisulfite sequencing PCR (BSP) assay showed that liver (D7, 7 days after birth) which expressed significantly lower dTERT mRNA had an obviously higher methylation level of S1 compared with small intestine (D7) or liver (E19). Quantitative real-time PCR analysis revealed that the expression of DNA methyltransferase DNMT1 in liver (D7) was significantly higher than that in small intestine (D7) or in liver (E19). In vitro, dTERT expression was upregulated and the methylation status of S1 decreased in both duck embryonic fibroblasts and small intestinal epithelial cells following treatment with the demethylation reagent, 5-aza-2'-deoxycytidine (5-aza-dC), further suggesting that dTERT is epigenetically regulated by DNA methylation. This work lays a solid foundation for further study of TERT function and regulation in avian species.
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Affiliation(s)
- Taoyan Yuan
- College of Animal Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China.,Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, 145 Shiqiao Road, Hangzhou 310021, China
| | - Wanqiu Zhao
- College of Animal Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China.,Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, 145 Shiqiao Road, Hangzhou 310021, China
| | - Yifan Niu
- College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, Henan, China
| | - Yan Fu
- College of Animal Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China
| | - Lizhi Lu
- Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, 145 Shiqiao Road, Hangzhou 310021, China.,Key Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture of China, Hangzhou 310021, Zhejiang, China
| | - Dong Niu
- College of Animal Science and Technology, Zhejiang A&F University
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45
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Chen X, Tang WJ, Shi JB, Liu MM, Liu XH. Therapeutic strategies for targeting telomerase in cancer. Med Res Rev 2019; 40:532-585. [PMID: 31361345 DOI: 10.1002/med.21626] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2019] [Revised: 07/12/2019] [Accepted: 07/16/2019] [Indexed: 12/13/2022]
Abstract
Telomere and telomerase play important roles in abnormal cell proliferation, metastasis, stem cell maintenance, and immortalization in various cancers. Therefore, designing of drugs targeting telomerase and telomere is of great significance. Over the past two decades, considerable knowledge regarding telomere and telomerase has been accumulated, which provides theoretical support for the design of therapeutic strategies such as telomere elongation. Therefore, the development of telomere-based therapies such as nucleoside analogs, non-nucleoside small molecules, antisense technology, ribozymes, and dominant negative human telomerase reverse transcriptase are being prioritized for eradicating a majority of tumors. While the benefits of telomere-based therapies are obvious, there is a need to address the limitations of various therapeutic strategies to improve the possibility of clinical applications. In this study, current knowledge of telomere and telomerase is discussed, and therapeutic strategies based on recent research are reviewed.
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Affiliation(s)
- Xing Chen
- School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Medical University, Hefei, People's Republic of China
| | - Wen-Jian Tang
- School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Medical University, Hefei, People's Republic of China
| | - Jing Bo Shi
- School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Medical University, Hefei, People's Republic of China
| | - Ming Ming Liu
- School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Medical University, Hefei, People's Republic of China
| | - Xin-Hua Liu
- School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Medical University, Hefei, People's Republic of China
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46
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Petrova OA, Mantsyzov AB, Rodina EV, Efimov SV, Hackenberg C, Hakanpää J, Klochkov VV, Lebedev AA, Chugunova AA, Malyavko AN, Zatsepin TS, Mishin AV, Zvereva MI, Lamzin VS, Dontsova OA, Polshakov VI. Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase. Nucleic Acids Res 2019; 46:1525-1540. [PMID: 29294091 PMCID: PMC5814841 DOI: 10.1093/nar/gkx1275] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2017] [Accepted: 12/19/2017] [Indexed: 12/19/2022] Open
Abstract
The elongation of single-stranded DNA repeats at the 3′-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids—telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the ‘fork’ at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.
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Affiliation(s)
- Olga A Petrova
- A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow 119991, Russia
| | - Alexey B Mantsyzov
- Centre for Magnetic Tomography and Spectroscopy, Faculty of Fundamental Medicine, M.V. Lomonosov Moscow State University, Moscow 119991, Russia
| | - Elena V Rodina
- Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia
| | - Sergey V Efimov
- NMR Laboratory, Institute of Physics, Kazan Federal University, 18 Kremlevskaya, Kazan 420008, Russia
| | - Claudia Hackenberg
- European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany
| | - Johanna Hakanpää
- European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany
| | - Vladimir V Klochkov
- NMR Laboratory, Institute of Physics, Kazan Federal University, 18 Kremlevskaya, Kazan 420008, Russia
| | - Andrej A Lebedev
- Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK
| | - Anastasia A Chugunova
- Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia.,Skolkovo Institute of Science and Technology, Moscow, Russia
| | - Alexander N Malyavko
- Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia.,Skolkovo Institute of Science and Technology, Moscow, Russia
| | - Timofei S Zatsepin
- Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia.,Skolkovo Institute of Science and Technology, Moscow, Russia
| | - Alexey V Mishin
- Laboratory for Structural Biology of GPCRs, Moscow Institute of Physics and Technology, Dolgoprudny, Russia
| | - Maria I Zvereva
- Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia
| | - Victor S Lamzin
- European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany
| | - Olga A Dontsova
- A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow 119991, Russia.,Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia.,Skolkovo Institute of Science and Technology, Moscow, Russia
| | - Vladimir I Polshakov
- Centre for Magnetic Tomography and Spectroscopy, Faculty of Fundamental Medicine, M.V. Lomonosov Moscow State University, Moscow 119991, Russia
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47
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Structural, functional, and stability change predictions in human telomerase upon specific point mutations. Sci Rep 2019; 9:8707. [PMID: 31213647 PMCID: PMC6581908 DOI: 10.1038/s41598-019-45206-y] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Accepted: 06/03/2019] [Indexed: 11/29/2022] Open
Abstract
Overexpression of telomerase is one of the hallmarks of human cancer. Telomerase is important for maintaining the integrity of the ends of chromosomes, which are called telomeres. A growing number of human disease syndromes are associated with organ failure caused by mutations in telomerase (hTERT or hTR). Mutations in telomerase lead to telomere shortening by decreasing the stability of the telomerase complex, reducing its accumulation, or directly affecting its enzymatic activity. In this work, potential human telomerase mutations were identified by a systematic computational approach. Moreover, molecular docking methods were used to predict the effects of these mutations on the affinity of certain ligands (C_9i, C_9k, 16A, and NSC749234). The C_9k inhibitor had the best binding affinity for wild-type (WT) telomerase. Moreover, C_9i and C_9k had improved interactions with human telomerase in most of the mutant models. The R631 and Y717 residues of WT telomerase formed interactions with all studied ligands and these interactions were also commonly found in most of the mutant models. Residues forming stable interactions with ligands in molecular dynamics (MD) were traced, and the MD simulations showed that the C_9k ligand formed different conformations with WT telomerase than the C_9i ligand.
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48
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Smathers CM, Robart AR. The mechanism of splicing as told by group II introns: Ancestors of the spliceosome. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2019; 1862:194390. [PMID: 31202783 DOI: 10.1016/j.bbagrm.2019.06.001] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/13/2019] [Accepted: 06/10/2019] [Indexed: 12/31/2022]
Abstract
Spliceosomal introns and self-splicing group II introns share a common mechanism of intron splicing where two sequential transesterification reactions remove intron lariats and ligate exons. The recent revolution in cryo-electron microscopy (cryo-EM) has allowed visualization of the spliceosome's ribozyme core. Comparison of these cryo-EM structures to recent group II intron crystal structures presents an opportunity to draw parallels between the RNA active site, substrate positioning, and product formation in these two model systems of intron splicing. In addition to shared RNA architectural features, structural similarity between group II intron encoded proteins (IEPs) and the integral spliceosomal protein Prp8 further support a shared catalytic core. These mechanistic and structural similarities support the long-held assertion that group II introns and the eukaryotic spliceosome have a common evolutionary origin. In this review, we discuss how recent structural insights into group II introns and the spliceosome facilitate the chemistry of splicing, highlight similarities between the two systems, and discuss their likely evolutionary connections. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.
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Affiliation(s)
- Claire M Smathers
- Department of Biochemistry, West Virginia University, Morgantown, WV, United States of America
| | - Aaron R Robart
- Department of Biochemistry, West Virginia University, Morgantown, WV, United States of America.
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49
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Nguyen THD, Collins K, Nogales E. Telomerase structures and regulation: shedding light on the chromosome end. Curr Opin Struct Biol 2019; 55:185-193. [PMID: 31202023 DOI: 10.1016/j.sbi.2019.04.009] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Revised: 03/28/2019] [Accepted: 04/29/2019] [Indexed: 02/06/2023]
Abstract
During genome replication, telomerase adds repeats to the ends of chromosomes to balance the loss of telomeric DNA. The regulation of telomerase activity is of medical relevance, as it has been implicated in human diseases such as cancer, as well as in aging. Until recently, structural information on this enzyme that would facilitate its clinical manipulation had been lacking due to telomerase very low abundance in cells. Recent cryo-EM structures of both the human and Tetrahymena thermophila telomerases have provided a picture of both the shared catalytic core of telomerase and its interaction with species-specific factors that play different roles in telomerase RNP assembly and function. We discuss also progress toward an understanding of telomerase RNP biogenesis and telomere recruitment from recent studies.
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Affiliation(s)
- Thi Hoang Duong Nguyen
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biology (QB3), University of California, Berkeley, CA 94720, USA; Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Miller Institute for Basic Research in Science, University of California, Berkeley, CA 94720, USA.
| | - Kathleen Collins
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biology (QB3), University of California, Berkeley, CA 94720, USA
| | - Eva Nogales
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biology (QB3), University of California, Berkeley, CA 94720, USA; Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA
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50
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Hernandez-Sanchez W, Huang W, Plucinsky B, Garcia-Vazquez N, Robinson NJ, Schiemann WP, Berdis AJ, Skordalakes E, Taylor DJ. A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity. PLoS Biol 2019; 17:e3000204. [PMID: 30951520 PMCID: PMC6469803 DOI: 10.1371/journal.pbio.3000204] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2018] [Revised: 04/17/2019] [Accepted: 03/14/2019] [Indexed: 12/13/2022] Open
Abstract
Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.
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Affiliation(s)
| | - Wei Huang
- Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States of America
| | - Brian Plucinsky
- The Wistar Institute Gene Expression and Regulation Program, Philadelphia, Pennsylvania, United States of America
| | - Nelson Garcia-Vazquez
- Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States of America
| | - Nathaniel J. Robinson
- Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States of America
| | - William P. Schiemann
- Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio, United States of America
| | - Anthony J. Berdis
- Department of Chemistry, Cleveland State University, Cleveland, Ohio, United States of America
| | - Emmanuel Skordalakes
- The Wistar Institute Gene Expression and Regulation Program, Philadelphia, Pennsylvania, United States of America
| | - Derek J. Taylor
- Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States of America
- Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, United States of America
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