Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show ...that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
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► Nascent RNA forms hybrids with underwound DNA upstream of elongating Pol II ► Single-stranded DNA so formed is prone to damage which results in genome instability ► Sen1 helicase acts to remove R loops by resolving RNA:DNA hybrids ► Sen1 function in Pol II elongation and termination may relate to R loop resolution
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
R loops have positive physiological roles, but they can also be deleterious by causing genome instability, and the mechanisms for this are unknown. Here we identified yeast histone H3 and H4 ...mutations that facilitate R loops but do not cause instability. R loops containing single-stranded DNA (ssDNA), versus RNA-DNA hybrids alone, were demonstrated using ssDNA-specific human AID and bisulfite. Notably, they are similar size regardless of whether or not they induce genome instability. Contrary to mutants causing R loop-mediated instability, these histone mutants do not accumulate H3 serine-10 phosphate (H3S10-P). We propose a two-step mechanism in which, first, an altered chromatin facilitates R loops, and second, chromatin is modified, including H3S10-P, as a requisite for compromising genome integrity. Consistently, these histone mutations suppress the high H3S10 phosphorylation and genomic instability of hpr1 and sen1 mutants. Therefore, contrary to what was previously believed, R loops do not cause genome instability by themselves.
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•Histones represent a first barrier to R loop formation•R loops per se do not cause genetic instability•H3 serine-10 phosphorylation is required for R loop-mediated genetic instability•Histone mutants unable to phosphorylate H3S10 suppress hpr1 genome instability
R loops can cause genome instability. However, García-Pichardo et al. identify histone H3/H4 mutants that demonstrate that R loops alone are not enough. Instead, further chromatin modifications, including H3 serine-10 phosphorylation, are necessary. They conclude that once R loops form, an additional chromatin modification step is required for genome instability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are ...those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.
DNA double-strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at ...transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with the recombinational repair, we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in
. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as
and
, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription and need to be removed to allow recombinational repair, rather than playing a positive role.
Transcription is a source of genome instability that stimulates mutation and recombination. Part of the damage produced by transcription is mediated by R-loops, non-B DNA structures that normally ...form by the re-annealing of the nascent RNA with the template DNA outside the catalytic center of the RNA polymerase, displacing the non-template strand. Recent discoveries have revealed that R-loops might not be harmful by themselves. Instead, chromatin compaction triggered by these structures seems necessary, as deduced from the histone modifications frequently found associated with harmful R-loops. Remarkably, hybrids may also become harmful if stabilized by specific RNA binding proteins, one example of which is the yeast Yra1. We discuss here the possible mechanisms by which cells may stabilize R-loops and the consequences on transcription-replication conflicts and telomere homeostasis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and ...required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA-DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA-DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA-DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.
The study by Tan-Wong et al. (2019) in this issue of Molecular Cell reveals a capacity of R-loops to promote antisense transcription expanding our view of the features that a DNA region may have to ...act as a promoter.
The study by Tan-Wong et al. (2019) in this issue of Molecular Cell reveals a capacity of R-loops to promote antisense transcription expanding our view of the features that a DNA region may have to act as a promoter.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Eukaryotic gene expression is a multilayer process covering transcription to post-translational protein modifications. As the nascent pre-mRNA emerges from the RNA polymerase II (RNAPII), it is ...packed in a messenger ribonucleoparticle (mRNP) whose optimal configuration is critical for the normal pre-mRNA processing and mRNA export, mRNA integrity as well as for transcription elongation efficiency. The interplay between transcription and mRNP formation feeds forward and backward and involves a number of conserved factors, from THO to THSC/TREX-2, which in addition have a unique impact on transcription-dependent genome instability. Here we review our actual knowledge of the role that these factors play at the interface between transcription and mRNA export in the model organism
Saccharomyces cerevisiae.
► THO and THSC play different roles at transcription/mRNA export interface ► Aberrant mRNPs accumulate in THO mutants forming R-loops ► R-loops cause genome instability ► Defects in mRNP biogenesis hinder RNAPII transcription
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Coupling of transcription with mRNA processing and export has been shown to be relevant to efficient gene expression. A number of studies have determined that THO/TREX, a nuclear protein complex ...conserved from yeast to humans, plays an important role in mRNP biogenesis connecting transcription elongation, mRNA export and preventing genetic instability. Recent data indicates that THO could be relevant to different mRNA processing steps, including the 3′-end formation, transcript release and export. Novel connections of THO to proteins related to the splicing machinery, provide new views about possible functions of THO in mRNP biogenesis. In this review, we summarize the previous and new results concerning the impact of THO in transcription and its biological implications, with a special emphasis on the relationship with THSC/TREX-2 and other functionally related factors involved in mRNA biogenesis and export. The emerging picture presents THO as a dynamic complex interacting with the nascent RNA and with different factors connecting nuclear functions necessary for mRNP biogenesis with genome integrity, cellular homeostasis and development. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
► THO is a mRNP factor necessary for the formation of export-competent mRNAs. ► THO/TREX is recruited to all active RNAPII transcribed genes, accumulating towards the 3 ′end. ► THO and THSC/TREX-2 are two key mRNP with a role in the maintenance of genome integrity. ► THO is a dynamic factor transiently interacting with THSC/TREX-2, CIP29/Tho1 and related factors
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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