Biology subjects

Aiello, U.

Publications and source records attributed to Aiello, U..

4 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↗

bioRxiv2025

Mutational scanning by multiplexed genome editing of the essential transcription termination factor Nrd1.

Proteins operate through a few critical residues, yet most proteins remain uncharacterized at the deep molecular resolution, particularly within essential genes, where functional dissection is obstructed by lethality. Here, we establish a platform for mutational scanning of essential genes at their endogenous locus, combining a repressible complementation system with multiplexed CRISPR-based genome editing in budding yeast. Our approach provides a generalizable framework for dissecting essential protein function in vivo, expanding the capacity to map critical residues underlying essential cellular processes. We applied this strategy to NRD1, encoding an essential RNA Polymerase II (RNAPII) termination factor and performed a systematic alanine scanning with near-saturation coverage. We discovered novel and unexpected lethal mutations in the CTD-interacting domain (CID), thus revealing an unanticipated importance for this domain. Overall, our results demonstrate the power of our mutation scanning platform to map critical residues underlying essential cellular processes.

genetics↗

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↗