Harmful R-loops are prevented via different cell cycle-specific mechanisms

Martín-Alonso, Marta San; Soler-Oliva, María E.; García-Rubio, María L.; García‐Muse, Tatiana; Aguilera, Andrés

doi:10.1038/s41467-021-24737-x

Cited by 43 publications

(68 citation statements)

References 82 publications

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“…We observed RNA:DNA hybrid hyper-accumulation during this type of collisions in sen1 mutants and a minor increase of these structures in co-directional clashes. These results are consistent with the previously described dynamic of hybrid accumulation in sen1 mutants at some highly-expressed genes ( 24 ) and genome-wide ( 33 ). While our findings are not in contrast with the notion that R-loops tend to accumulate in both types of collisions ( 6 , 31 ), they do suggest that R-loops contribute to fork arrest only in head-on clashes.…”

Section: Discussionsupporting

confidence: 93%

“…In Sen1-depleted cells, head-on highly transcribed RNAPII genes accumulate R-loops ( 24 , 33 ) and form an impenetrable barrier to replication fork ( 34 ). The activation of dormant origins near the collision sites is a mechanism to rescue stalled replication forks, while the protection of replisome prevents the nuclease-mediated processing of R-loops at the fork ( 34 ).…”

Section: Introductionmentioning

confidence: 99%

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Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Zardoni

Nardini

Brambati

et al. 2021

Nucleic Acids Research

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Uncoordinated clashes between replication forks and transcription cause replication stress and genome instability, which are hallmarks of cancer and neurodegeneration. Here, we investigate the outcomes of head-on replication-transcription collisions, using as a model system budding yeast mutants for the helicase Sen1, the ortholog of human Senataxin. We found that RNA Polymerase II accumulates together with RNA:DNA hybrids at sites of head-on collisions. The replication fork and RNA Polymerase II are both arrested during the clash, leading to DNA damage and, in the long run, the inhibition of gene expression. The inactivation of RNA Polymerase II elongation factors, such as the HMG-like protein Spt2 and the DISF and PAF complexes, but not alterations in chromatin structure, allows replication fork progression through transcribed regions. Attenuation of RNA Polymerase II elongation rescues RNA:DNA hybrid accumulation and DNA damage sensitivity caused by the absence of Sen1, but not of RNase H proteins, suggesting that such enzymes counteract toxic RNA:DNA hybrids at different stages of the cell cycle with Sen1 mainly acting in replication. We suggest that the main obstacle to replication fork progression is the elongating RNA Polymerase II engaged in an R-loop, rather than RNA:DNA hybrids per se or hybrid-associated chromatin modifications.

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Zardoni

Nardini

Brambati

et al. 2021

Nucleic Acids Research

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“…RNAPII accumulation was also observed in rnh1𝚫 rnh201𝚫 cells suggesting that RNase H is required at this locus for efficient RNAPII release. Consistently, R-loops were detected, both by H-CRAC and DRIP (San Martin-Alonso et al, 2021;Wahba et al, 2016) immediately upstream of the paused polymerase (Figure S5B). Interestingly, we also noticed the occurrence of transcription upstream and antisense of RDN5 directed towards the close rARS (ARS1200) replication origin (Figure 5C), which was only observed in replicating cells and was also RNases H dependent.…”

Section: Roles Of Sen1 and Rnases H In Limiting Rnapii Transcription ...supporting

confidence: 62%

“…RNAPII CRAC is also shown for evaluating the R-loop signals relative to transcription. For the directional DRIP-seq (San Martin-Alonso et al, 2021), the H-CRAC and the RNAPII CRAC only the strand of the target gene is shown. B) Overlap of the genes containing the highest signals (levels higher than the mean plus one standard deviation) defined by the directional DRIP-seq (San Martin-Alonso et al, 2021) and by H-CRAC (Rnh1 or Rnh201).…”

Section: Resultsmentioning

confidence: 99%

Sen1 is a key regulator of transcription-driven conflicts

Aiello

Challal

Wentzinger

et al. 2022

Preprint

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Cellular homeostasis requires the coordination of several machineries concurrently engaged on the DNA. Wide-spread transcription can interfere with other processes and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase terminates non-coding transcription, but also interacts with the replisome and reportedly resolves genotoxic R-loops. Sen1 prevents genomic instability but how this relates to its molecular functions remains unclear. We generated high-resolution, genome-wide maps of transcription-dependent conflicts and R-loops using a Sen1 mutant that has lost interaction with the replisome but is termination proficient. We show that Sen1 removes RNA polymerase II at TRCs within genes and the rDNA, but also at sites of transcription-transcription conflicts under physiological conditions, thus qualifying as a "master regulator of conflicts". We demonstrate that genomic stability is only affected by Sen1 mutation when, in addition to its role at the replisome, termination of non-coding transcription or R-loop removal are additionally compromised.

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“…In previous studies, abolishment of either the THO or TRAMP complex has been shown to increase R-loop-associated genome instability [22, 36, 37, 51]. Recently, the THO complex was shown to prevent R-loop formation throughout the cell cycle whereas other factors such as Senataxin only resolve R-loops during S-phase [52]. We previously showed that R-loops form preferentially at expanded CAG tracts in vivo and that increasing R-loop stability by removing both RNase H1 and RNase H2 proteins using a rnh1Δrnh201Δ double mutant further increased CAG repeat fragility [7].…”

Section: Resultsmentioning

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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Harmful R-loops are prevented via different cell cycle-specific mechanisms

Cited by 43 publications

References 82 publications

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions

Sen1 is a key regulator of transcription-driven conflicts

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

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