Defining R-loop classes and their contributions to genome instability

Castillo-Guzman, Daisy; Chédin, Frédéric

doi:10.1016/j.dnarep.2021.103182

Cited by 62 publications

(49 citation statements)

References 115 publications

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“…RDProx relies on the HBD of RNaseH1 and is therefore inherently biased to the R-loops that are recognized and bound by RNaseH1. Recently, the existence of different classes of R-loops—promoter-paused R-loops and elongation-associated R-loops—that each display unique characteristics was proposed 57 , 58 . Promoter-paused R-loops are short R-loops frequently forming during promoter-proximal pausing of RNAPII 57 , 58 .…”

Section: Discussionmentioning

confidence: 99%

“…Recently, the existence of different classes of R-loops—promoter-paused R-loops and elongation-associated R-loops—that each display unique characteristics was proposed 57 , 58 . Promoter-paused R-loops are short R-loops frequently forming during promoter-proximal pausing of RNAPII 57 , 58 . R-loop mapping approaches based on RNaseH1 showed an enrichment of RNaseH1 at promoter-proximal sites 43 , 46 .…”

Section: Discussionmentioning

confidence: 99%

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R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

et al. 2021

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Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA–DNA hybrid with a displaced non-template DNA strand. We developed RNA–DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA–DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

et al. 2021

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“…While R-loop formation at gene promoters (transcription start sites or TSSs) appears to be the general consensus, various studies also suggest the broad distribution of R-loops in gene bodies, transcription termination sites (TTSs), and even intergenic regions. Because the locations of R-loops in different genomic regions would have dramatically distinct implications in R-loop biology, it is important to systematically determine whether different R-loop mapping strategies are of specific biases in capturing R-loops in different genomic regions or how individual strategies may each introduce different types of artifacts in different degrees (Castillo-Guzman and Chedin 2021; Belotserkovskii and Hanawalt 2022).…”

Section: Introductionmentioning

confidence: 99%

Systematic Evaluation of Different R-loop Mapping Methods: Achieving Consensus, Resolving Discrepancies and Uncovering Distinct Types of RNA:DNA Hybrids

Chen

Lim

Chen

et al. 2022

Preprint

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R-loop, a three-stranded nucleic acid structure, has been recognized to play pivotal roles in critical physiological and pathological processes. Multiple technologies have been developed to profile R-loops genome-wide, but the existing data suffer from major discrepancies on determining genuine R-loop localization and its biological functions. Here, we experimentally and computationally evaluate eight representative R-loop mapping technologies, and reveal inherent biases and artifacts of individual technologies as key sources of discrepancies. Analyzing signals detected with different R-loop mapping strategies, we note that genuine R-loops predominately form at gene promoter regions, whereas most signals in gene body likely result from structured RNAs as part of repeat-containing transcripts. Interestingly, our analysis also uncovers two classes of R-loops: The first class consists of typical R-loops where the single-stranded DNA binding protein RPA binds both the template and non-template strands. By contrast, the second class appears independent of Pol II-mediated transcription and is characterized by RPA binding only in the template strand. These two different classes of RNA:DNA hybrids in the genome suggest distinct biochemical activities involved in their formation and regulation. In sum, our findings will guide future use of suitable technology for specific experimental purposes and the interpretation of R-loop functions.

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“…Castillo-Guzman and Chédin recently defined two classes of R-loops, promoter-paused (Class I) and elongation-associated (Class II). The types of R-loops that accumulate at the CAG repeat in the absence of Thp2 may be similar to Class I R-loops that are shorter, less stable, and not readily detected by DRIP [55].…”

Section: Resultsmentioning

confidence: 99%

“…Because we could not directly detect an increase in R-loops at the CAG tract by DRIP in the thp2Δ mutant, but only detected their phenotypic consequence by the RNase H1 overexpression experiments, we hypothesize that they are transient and likely associated with the stalled RNAPII, rather than stable R-loops left after passage of the transcriptional machinery. It has been recently recognized that small R-loops (60 bp or less) associated with paused RNAPII are not detected by DRIP, which preferentially detects longer (>200 bp), more stable R-loops [55]. Based on the role of THO in binding nascent RNA to direct it to the nuclear pore complex for export, it is probable that the unbound nascent RNA is more likely to re-anneal to the DNA template in this mutant, inhibiting RNAPII progression and causing an accumulation of RNAPII within the CAG tract (Fig.…”

Section: Discussionmentioning

confidence: 99%

THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

Brown

Fair

et al. 2021

Preprint

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Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2∆ mutants. In contrast, CAG fragility and TRCs in the trf4∆ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

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Defining R-loop classes and their contributions to genome instability

Cited by 62 publications

References 115 publications

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

Systematic Evaluation of Different R-loop Mapping Methods: Achieving Consensus, Resolving Discrepancies and Uncovering Distinct Types of RNA:DNA Hybrids

THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

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