Publicação
Transcription dynamics prevents RNA-mediated genomic instability through SRPK2-dependent DDX23 phosphorylation
| Resumo: | During transcription, RNA is synthesized by RNA polymerase II (RNA Pol II) using the information contained in the template DNA strand. RNA synthesis occurs inside the transcription bubble where the two strands of DNA are physically separated and the nascent transcript is hybridized to template strand through a ~8 base pair (bp) RNA-DNA hybrid. Latter, the molecular structure of the elongating RNA Pol II ensures that the nascent transcript leaves the transcription bubble physically separated from the template DNA. Therefore, it was widely believed that RNA-DNA hybrids were only transient byproducts of transcription. However, the past decade of research has shown those R-loops − RNA-DNA hybrids and a displaced single-stranded DNA−are abundant structures that play important roles in regulating gene expression and DNA recombination. Besides these physiological roles, R-loops also pose great threats to genome stability and should therefore be tightly regulated. Cells regulate the level of R-loops by employing enzymes like DNA/RNA helicases and nucleases to resolve the RNA moiety of R-loops. Additionally, various RNA processing factors are also known to prevent R-loop formation by sequestering the nascent RNA, thus preventing it from re-annealing to the template DNA. However, the signalling cascades and molecular switches that lead to the mobilization and activation of R-loop suppressors are essentially unknown. The activity of several RNA processing factors is regulated through phosphorylation, which in many cases is driven by serine/arginine protein kinases (SRPK) 1 and 2. These kinases phosphorylate the arginine-serine (RS) domain of various RNA processing factors classified as SR-proteins. Phosphorylation/de phosphorylation cycles regulate the function of SR-proteins and the timely binding to cis regulatory elements on the nascent RNA. However, the roles of both SRPK1 and SRPK2 in R-loop metabolism and genome stability have not yet been investigated. Herein, we identify a new role for RPK2 in preventing genomic instability through a mechanism involving the suppression of R-loops. We show that phosphorylation of the DEAD box helicase-DDX23 is necessary and sufficient to restore the genome stability in SRPK2-deficient cells through a process that requires its helicase activity. DDX23 is part of the spliceosomal U5 small nuclear ribonucleoprotein complex (U5 snRNP). We found that the role of DDX23 in suppressing R-loops does not require a functional U5 snRNP as depletion of either PRP8 or PRP6, core components of the U5 1 2 snRNP, does not drive genomic instability. Altogether, we found that phosphorylation of DDX23 by SRPK2 is a crucial event to maintain cellular R-loop levels, failure of which severely compromises genome integrity. Co-transcriptional R-loops impact on RNA Pol II transcription dynamics. For instance, they are involved in promoter-proximal pausing and promote efficien transcription termination by slowing down RNA Pol II facilitating the timely recruitment of termination factors. Here we describe a new pathway that employs oscillations in RNA Pol II dynamics as a molecular sensor to signal the location of R-loops and nucleate SRPK2-dependent DDX23 phosphorylation. This may constitute a new genome caretaker mechanism that would operate to ward off against tumour-driving DNA damage. Supporting this view, we observed that loss of DDX23 is a prevalent feature of adenoid cystic carcinoma, an aggressive salivary gland cancer with very limited treatment options mostly due to our incomplete understanding of its genomic foundations and molecular basis. |
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| Autores principais: | Sridhara, Sree Rama Chaitanya |
| Assunto: | RNA Transcrição genética Fosforilação Factores de processamento de serina-arginina RNA helicases DEAD-box Teses de doutoramento - 2017 |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | During transcription, RNA is synthesized by RNA polymerase II (RNA Pol II) using the information contained in the template DNA strand. RNA synthesis occurs inside the transcription bubble where the two strands of DNA are physically separated and the nascent transcript is hybridized to template strand through a ~8 base pair (bp) RNA-DNA hybrid. Latter, the molecular structure of the elongating RNA Pol II ensures that the nascent transcript leaves the transcription bubble physically separated from the template DNA. Therefore, it was widely believed that RNA-DNA hybrids were only transient byproducts of transcription. However, the past decade of research has shown those R-loops − RNA-DNA hybrids and a displaced single-stranded DNA−are abundant structures that play important roles in regulating gene expression and DNA recombination. Besides these physiological roles, R-loops also pose great threats to genome stability and should therefore be tightly regulated. Cells regulate the level of R-loops by employing enzymes like DNA/RNA helicases and nucleases to resolve the RNA moiety of R-loops. Additionally, various RNA processing factors are also known to prevent R-loop formation by sequestering the nascent RNA, thus preventing it from re-annealing to the template DNA. However, the signalling cascades and molecular switches that lead to the mobilization and activation of R-loop suppressors are essentially unknown. The activity of several RNA processing factors is regulated through phosphorylation, which in many cases is driven by serine/arginine protein kinases (SRPK) 1 and 2. These kinases phosphorylate the arginine-serine (RS) domain of various RNA processing factors classified as SR-proteins. Phosphorylation/de phosphorylation cycles regulate the function of SR-proteins and the timely binding to cis regulatory elements on the nascent RNA. However, the roles of both SRPK1 and SRPK2 in R-loop metabolism and genome stability have not yet been investigated. Herein, we identify a new role for RPK2 in preventing genomic instability through a mechanism involving the suppression of R-loops. We show that phosphorylation of the DEAD box helicase-DDX23 is necessary and sufficient to restore the genome stability in SRPK2-deficient cells through a process that requires its helicase activity. DDX23 is part of the spliceosomal U5 small nuclear ribonucleoprotein complex (U5 snRNP). We found that the role of DDX23 in suppressing R-loops does not require a functional U5 snRNP as depletion of either PRP8 or PRP6, core components of the U5 1 2 snRNP, does not drive genomic instability. Altogether, we found that phosphorylation of DDX23 by SRPK2 is a crucial event to maintain cellular R-loop levels, failure of which severely compromises genome integrity. Co-transcriptional R-loops impact on RNA Pol II transcription dynamics. For instance, they are involved in promoter-proximal pausing and promote efficien transcription termination by slowing down RNA Pol II facilitating the timely recruitment of termination factors. Here we describe a new pathway that employs oscillations in RNA Pol II dynamics as a molecular sensor to signal the location of R-loops and nucleate SRPK2-dependent DDX23 phosphorylation. This may constitute a new genome caretaker mechanism that would operate to ward off against tumour-driving DNA damage. Supporting this view, we observed that loss of DDX23 is a prevalent feature of adenoid cystic carcinoma, an aggressive salivary gland cancer with very limited treatment options mostly due to our incomplete understanding of its genomic foundations and molecular basis. |
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