Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis

Hage, Aziz El; French, Sarah L.; Beyer, Ann L.; Tollervey, David

doi:10.1101/gad.573310

Cited by 375 publications

(469 citation statements)

References 49 publications

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“…4A, Left and Fig. S4A), and consistent with previous observations (19), growth of r1Δ r2Δ mutants was inhibited in the absence of Top1 (Fig. S4A).…”

Section: Depletion Of Top1 In the Absence Of Rnase H Leads To Unschedsupporting

confidence: 79%

“…Highly transcribed ribosomal genes have been shown to favor R-loop formation in cells lacking both RNase H and Top1 activities (19). Here, by taking advantage of R-loop promoting conditions we potentiate the formation of DNA double-strand breaks (DSBs) and detect origin-independent replication intermediates (RI) within the transcribed 35S rRNA genes.…”

mentioning

confidence: 99%

“…11). To do so, cells catalyze the relaxation of supercoiled DNA by type I topoisomerases (12)(13)(14)(15), thus preventing replication fork reversal (16), DNA overwinding with the potential to block replication fork progression (17), DNA unwinding (18), or R-loop-mediated blocks of ribosomal RNA synthesis (19). Other enzymatic activities involved in R-loop processing include ribonuclease H (RNase H) activities, DNA-RNA helicases, such as Sen1/senataxin (20,21), or Ataxin-2 RNA-binding protein Pbp1 (10).…”

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confidence: 99%

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Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Stuckey

García‐Rodríguez

Aguilera

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop-mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis.D uring transcription, DNA acts as a template for the synthesis of the nascent RNA. RNA synthesis is accompanied by the generation of positive and negative DNA supercoiling in front of and behind the transcription machinery, respectively (1). Unwinding of the DNA double helix by negative supercoiling may allow the RNA to hybridize to its DNA template behind the elongating RNA polymerase, leading to R-loops (2). Other elements that could potentiate R-loop accumulation include RNA: DNA hybrid-facilitating DNA sequences, such as G-quadruplex structures (3) or nicks in the nontemplate DNA strand (4).Eukaryotic cells need to control R-loop formation to avoid replication impairment, genome instability, and life span shortening mediated by such intermediates (5-10; reviewed in ref. 11). To do so, cells catalyze the relaxation of supercoiled DNA by type I topoisomerases (12-15), thus preventing replication fork reversal (16), DNA overwinding with the potential to block replication fork progression (17), DNA unwinding (18), or R-loop-mediated blocks of ribosomal RNA synthesis (19). Other enzymatic activities involved in R-loop processing include ribonuclease H (RNase H) activities, DNA-RNA helicases, such as Sen1/senataxin (20, 21), or Ataxin-2 RNA-binding protein Pbp1 (10). The ribonuclease activity of Saccharomyces cerevisiae RNases H1 and H2 specifically cleaves the RNA moiety of the RNA:DNA hybrid structure (22), whereas RNase H2 and topoisomerase 1 (Top1) can also process ribonucleotides in duplex DNA (23,24).Notably, R-loops are required to initiate mitochondrial DNA replication (25) and pioneering studies connected R-loops to origin-independent replication in prokaryotic systems (26,27). For example, DnaA-dependent initiation of DNA replication at the Escherichia coli oriC replication origin can be overcome in the absence of RNase H1 (28, 29). As a consequence, rnhA mutants can survive complete inactivation of oriC by transcriptiondependent activation of so-called oriK sites (30, 31), although candidate oriK sites have been identified only recently (32). Additional evidence for R-loop-primed replication was given by the observation that rnhA mutants are prone to an increase in mutation and DNA amplification events if origin activity is suppressed. These events required remov...

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“…4A, Left and Fig. S4A), and consistent with previous observations (19), growth of r1Δ r2Δ mutants was inhibited in the absence of Top1 (Fig. S4A).…”

Section: Depletion Of Top1 In the Absence Of Rnase H Leads To Unschedsupporting

confidence: 79%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Stuckey

García‐Rodríguez

Aguilera

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…These findings clearly demonstrate the essential role of post-initiation steps in the regulation of rRNA synthesis by growth factors. Certainly, work in yeast revealed a complex interplay between rRNA production, assembly, cleavage, and folding that occurs during elongation [36,51]. Uncovering how elongation is regulated and how it is coupled to rRNA assembly will lead to major advances towards understanding the mechanisms of rRNA production [49].…”

Section: Accepted Manuscript 18mentioning

confidence: 99%

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Hung

Lesmana

Peck

et al. 2017

Gene

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, Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2016Gene( ), doi: 10.1016Gene( /j.gene.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T 3 AbstractTranscription of the ribosomal RNA genes (rDNA) by RNA Polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth.However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labelling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation.

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“…It is tempting to speculate that at distally paused genes, the enhancer might bring in factors capable of resolving R-loops or other physical constraints (e.g., supercoiling). To this end, inhibition or down-regulation of type I topoisomerases leads to an accumulation of R-loops at the MYC locus (Yang et al 2014) or ribosomal RNA genes (El Hage et al 2010;Marinello et al 2013). How enhancers might be selectively directed to the promoter versus the CGI edge is unclear, but recent evidence showing that a substantial fraction of long-range looping interactions is stable across cell types and developmental windows and precedes signal-induced gene activation (Jin et al 2013;Ghavi-Helm et al 2014) suggests that at least one component of enhancer activity (looping) may be linked to underlying sequence features.…”

Section: Org Downloaded Frommentioning

confidence: 99%

GC skew defines distinct RNA polymerase pause sites in CpG island promoters

2015

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CpG islands (CGIs) are associated with over half of human gene promoters and are characterized by a unique chromatin environment and high levels of bidirectional transcriptional activity relative to surrounding genomic regions, suggesting that RNA polymerase (Pol II) progression past the CGI boundaries is restricted. Here we describe a novel transcriptional regulatory step wherein Pol II encounters an additional barrier to elongation distinct from the promoter-proximal pause and occurring at the downstream boundary of the CGI domain. For most CGI-associated promoters, Pol II exhibits a dominant pause at either the promoter-proximal or this distal site that correlates, both in position and in intensity, with local regions of high GC skew, a sequence feature known to form unique secondary structures. Upon signal-induced gene activation, long-range enhancer contacts at the dominant pause site are selectively enhanced, suggesting a new role for enhancers at the downstream pause. These data point to an additional level of control over transcriptional output at a subset of CGI-associated genes that is linked to DNA sequence and the integrity of the CGI domain.

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Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis

Cited by 375 publications

References 49 publications

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

GC skew defines distinct RNA polymerase pause sites in CpG island promoters

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