TY - JOUR
T1 - Deficiency in classical nonhomologous end-joining-mediated repair of transcribed genes is linked to SCA3 pathogenesis
AU - Chakraborty, Anirban
AU - Tapryal, Nisha
AU - Venkova, Tatiana
AU - Mitra, Joy
AU - Vasquez, Velmarini
AU - Sarker, Altaf H.
AU - Duarte-Silva, Sara
AU - Huai, Weihan
AU - Ashizawa, Tetsuo
AU - Ghosh, Gourisankar
AU - MacIel, Patricia
AU - Sarkar, Partha S.
AU - Hegde, Muralidhar L.
AU - Chen, Xu
AU - Hazra, Tapas K.
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Sahn-ho Kim, Lawrence Berkeley National Laboratory, for providing critical help and necessary reagents for large-scale fractionations of HeLa-S3 cell nuclear extract by gel filtration; Dr. Sankar Mitra for critically reading the manuscript; and Dr. Sarah Toombs Smith, Editor in the Life Sciences, University of Texas Medical Branch (UTMB) for editing this manuscript. We thank the Michigan Brain Bank (5P30 AG053760 University of Michigan Alzheimer’s Disease Core Center) for providing us with the tissue of postmortem SCA3 patients and their age-matched controls. This work was supported by National Institute of Health Grants 2R01 NS073976 (to T.K.H.), R01 NS096305 (to P.S.S. and T.K.H.), and R01 NS088645 (to M.L.H.); University of California Tobacco Related Disease Research Program Grant 26IR-0017 (to A.H.S.) and P30 ES 06676 (to the National Institute on Environmental Health Sciences Center grant to Cell Biology and Molecular Genomics Core of the University of Texas Medical Branch). Research at the Life and Health Sciences Research Institute was supported by the European Regional Development Fund (FEDER), through the Competitiveness Internationalization Operational Programme (POCI), and by national funds, through the Foundation for Science and Technology, under the scope of the projects POCI-01-0145-FEDER-031987 and POCI-01-0145-FEDER-029056, and by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement (NORTE-01-0145-FEDER-000013).
Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.
PY - 2020/4/7
Y1 - 2020/4/7
N2 - Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.
AB - Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.
KW - ATXN3
KW - DNA double-strand break repair
KW - PNKP
KW - RNA-templated TC-NHEJ
KW - Spinocerebellar ataxia type-3
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U2 - 10.1073/pnas.1917280117
DO - 10.1073/pnas.1917280117
M3 - Article
C2 - 32205441
AN - SCOPUS:85083089024
SN - 0027-8424
VL - 117
SP - 8154
EP - 8165
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 14
ER -