Biology subjects

Libri, D.

Publications and source records attributed to Libri, D..

11 recordsLinked to original sources

bioRxiv2026

DNA:RNA hybrid mutational landscapes reveal a common route to geneticinstability in evolving genomes

Pervasive DNA:RNA hybrids are recognized as pathological sources of genome instability, yet how their genotoxicity shapes mutational landscapes and genome evolution remains poorly understood. Here, we define the mutational footprint of hybrids by integrating genome-wide mapping, long-term mutation accumulation experiments, and analyses of genetic diversity across thousands of natural yeast genomes. We uncover signatures for embedded ribonucleotides and genic R-loops, together with a composite mutational pattern shared across natural populations and experimental evolution. Using reporters designed to dissect the mechanisms underlying genetic alterations, we find that they arise primarily from targeting by MutL{gamma}-dependent mismatch repair followed by Rad52-mediated recombination. Although hybrids form dynamically across the genome, their genotoxicity is strongly skewed toward repetitive regions susceptible to these error-prone pathways, including microsatellites and retroelements. Our findings reveal a common mechanism of hybrid-associated mutagenesis that can shape genetic variation in evolving genomes.

molecular biology↗

bioRxiv2022

The S. cerevisiae m6A-reader Pho92 impacts meiotic recombination by controlling key methylated transcripts

N6-methyladenosine (m6A), the most abundant internal modification of eukaryotic mRNAs, participates in the post-transcriptional control of gene expression. In Saccharomyces cerevisiae, m6A is only found during meiosis. Although the deletion of the m6A- methyltransferase Ime4 impairs this process, the molecular impact of m6A on gene expression remains ill defined. Here we investigated the function of the budding yeast m6A reader Pho92. We found that Pho92 is specifically expressed during meiosis and impacts meiotic progression. We used high-throughput RNA sequencing and mapping of Pho92-binding sites following UV-crosslinking to show that Pho92 is recruited to specific mRNAs in an m6A-dependent manner during the meiotic prophase, preceding their down-regulation. Strikingly, point mutations altering m6A sites in mRNAs targeted by Pho92 are sufficient to delay their down-regulation and, in one case, to impact meiotic progression. Altogether, our results indicate that Pho92 facilitate the meiotic progression by accelerating the down-regulation of timely-regulated mRNAs during meiotic recombination.

molecular biology↗

bioRxiv2022

Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly-dT termination signals

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on poly-thymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryo-EM structures of the yeast Pol III PTC and complementary functional states at 2.7-3.9 [A] resolution. Pol III recognizes the poly-dT termination signal with subunit C128 that forms a hydrogen-bond network with the NT-strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly-dT signals.

biochemistry↗

bioRxiv2022

Sen1 is a master regulator of transcription-driven conflicts

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 "key 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.

genomics↗

bioRxiv2021

An integrated model for termination of RNA polymerase III transcription

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.

genomics↗

bioRxiv2020

Xrn1 influences RNA pol II-dependent transcription elongation rates across the yeast genome and this control is particularly relevant for late elongation of regulatory genes

mRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5 exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3 was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favoring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.

genomics↗

bioRxiv2019

Degradation of non-coding RNAs promotes recycling of termination factors at sites of transcription

A large share of the non-coding transcriptome in yeast is controlled by the Nrd1-Nab3-Sen1 (NNS) complex, which promotes transcription termination of non-coding RNA (ncRNA) genes, and by the nuclear exosome, which limits the steady state levels of the transcripts produced. How unconstrained ncRNA levels impact RNA metabolism and gene expression are longstanding and important question. Here we show that degradation of ncRNAs by the exosome is required for freeing Nrd1 and Nab3 from the released transcript after termination. In exosome mutants, these factors are sequestered by ncRNAs and cannot be efficiently recycled to sites of transcription, inducing termination defects at NNS targets. ncRNA-dependent, genome-wide termination defects can be recapitulated by the expression of a degradation-resistant, circular RNA containing a natural NNS target in exosome proficient cells. Our results have important implications for the mechanism of termination, the general impact of ncRNAs abundance and the importance of nuclear ncRNA degradation.

molecular biology↗

bioRxiv2019

Opposing chromatin remodelers control transcription initiation frequency and start site selection

Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome displacement activity following rapid remodeler depletion, we investigate the interplay between these enzymes and their impact on transcription in budding yeast. We show that many promoters are acted upon by multiple CRs that operate either cooperatively or in opposition to position the key transcription start site-associated +1 nucleosome. Functional assays suggest that +1 nucleosome positioning often reflects a trade-off between maximizing RNA Polymerase II recruitment and minimizing transcription initiation at incorrect sites. Finally, we show that nucleosome movement following CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity +1 nucleosomes maintain their positions. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.

molecular biology↗

bioRxiv2018

Termination of non-coding transcription in yeast relies on both a CTD-interaction domain and a CTD-mimic in Sen1

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a risk that needs to be controlled to prevent the perturbation of gene expression. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy terminal domain (CTD) of RNA polymerase II and characterize structurally its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires the recognition of the CTD by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote Sen1 initial recruitment but rather the capacity of Sen1 to induce the release of paused RNAPII from the DNA. Our results shed light onto the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

genetics↗

bioRxiv2018

Pervasive Transcription Fine-tunes Replication Origin Activity

RNA polymerase (RNAPII) transcription occurs pervasively, which raises the important question of its functional impact on other DNA-associated processes, including replication. In budding yeast, replication originates from Autonomously Replicating Sequences (ARSs), generally located in intergenic regions. The influence of transcription on ARSs function has been studied for decades, but these earlier studies have necessarily neglected the role of non-annotated transcription. We studied the relationships between pervasive transcription and replication origin activity using high-resolution transcription maps. We show that ARSs alter the pervasive transcription landscape by pausing and terminating neighboring RNAPII transcription, thus limiting the occurrence of pervasive transcription within origins. We provide evidence that quasi-symmetrical binding of the ORC complex to ARS borders is responsible for pausing/termination. We also show that low, physiological levels of pervasive transcription impact the function of replication origins. Overall, our results have important implications for understanding the impact of genomic location on origin function.

genetics↗

bioRxiv2018

General Regulatory Factors control the fidelity of transcription by restricting non-coding and ectopic initiation

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other General Regulatory Factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, profoundly altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We show that Rap1 sterically constrains nucleosomes as its mere binding to the DNA can be sufficient for restoration normal nucleosome positioning, transcription initiation and gene expression. These results demonstrate an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, redefining current models of their function. They have general implications for the mechanism of transcription initiation and the control of gene expression.\n\nHIGHLIGHTSO_LIRap1, Abf1 and Reb1 control the fidelity of transcription initiation and suppress pervasive transcription\nC_LIO_LIWidespread ectopic transcription initiation in Rap1-deficient cells induces variegated alterations in gene expression\nC_LIO_LIAltered nucleosome positioning in GRFs-defective cells correlate with ectopic transcription initiation.\nC_LIO_LIRap1 controls nucleosomes positioning and transcription initiation at least partially by a steric hindrance mechanism\nC_LI

genomics↗