Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability

Appanah, Rowin; Lones, Emma Claire; Aiello, Umberto; Libri, Domenico; Piccoli, Giacomo De

doi:10.1016/j.celrep.2020.01.087

Cited by 27 publications

(47 citation statements)

References 66 publications

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“…We expressed both SEN1 variants from the GAL1 promoter (pGAL1) because only overexpression of the sen1DNTD allele supports viability (Han et al, 2020). In agreement with previous reports (Appanah et al, 2020;Yüce and West, 2013), we detected an RNase-resistant interaction of Sen1 with its partners within the NNS-complex Nrd1 and Nab3, several replication and transcription-related factors, as well as with the three RNAPs. Strikingly, deletion of the NTD abolished the association of Sen1 with RNAPIII and most replication factors without markedly affecting other interactions.…”

Section: Resultssupporting

confidence: 88%

An integrated model for termination of RNA polymerase III transcription

Xie

Aiello

Clément

et al. 2021

Preprint

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RNA polymerase III (RNAPIII) synthesizes essential and abundant non-coding RNAs such as tRNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the non-template strand. Here we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

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Section: Resultssupporting

confidence: 88%

An integrated model for termination of RNA polymerase III transcription

Xie

Aiello

Clément

et al. 2021

Preprint

Self Cite

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“…R-loops are DNA-RNA hybrid structures and have been shown to form in the rDNA locus ( 32 ). RNase H1 specifically degrades R-loops ( 33 ) and has been shown to remove R-loops from the rDNA locus of budding yeast in vitro ( 34 , 35 ) and in vivo ( 36–38 ). We transformed a yeast strain containing Cdc14-GFP and Cbf5-mCherry with a vector control plasmid (pBL189) or with a plasmid expressing the RNase H1 gene Rnh1 (pBB39) on a high copy number plasmid ( 34 ) (Figure 10A and B ).…”

Section: Resultsmentioning

confidence: 99%

The rDNA is biomolecular condensate formed by polymer–polymer phase separation and is sequestered in the nucleolus by transcription and R-loops

Lawrimore

Kolbin

Stanton

et al. 2021

Nucleic Acids Research

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The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer–polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14’s nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA–binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA–DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer–polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus.

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“…7 ). Since Sen1 is a helicase involved in transcription termination, which has been shown to play a key role at RF encounters with TTSs 22 , 66 , and function in S-phase 62 , it may be crucial to remove such R-loops and to prevent major consequences of the RF blockage. Consistently, pausing at the TTS of highly expressed human genes containing R-loops prevents HO T-R conflicts 67 .…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.

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Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability

Cited by 27 publications

References 66 publications

An integrated model for termination of RNA polymerase III transcription

An integrated model for termination of RNA polymerase III transcription

The rDNA is biomolecular condensate formed by polymer–polymer phase separation and is sequestered in the nucleolus by transcription and R-loops

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

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