RESUMO
Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins.
Assuntos
Rearranjo Gênico/genética , Instabilidade Genômica/genética , Estruturas R-Loop/genética , Envelhecimento/genética , Aberrações Cromossômicas , Pontos de Quebra do Cromossomo , Quebras de DNA de Cadeia Dupla , Genômica/métodos , Modelos Genéticos , Mutação/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Telômero/genéticaRESUMO
The synthesis, processing and function of coding and non-coding RNA molecules and their interacting proteins has been the focus of a great deal of research that has boosted our understanding of key molecular pathways that underlie higher order events such as cell cycle control, development, innate immune response and the occurrence of genetic diseases. In this study, we have found that formamide preferentially weakens RNA related processes in vivo. Using a non-essential Schizosaccharomyces pombe gene deletion collection, we identify deleted loci that make cells sensitive to formamide. Sensitive deletions are significantly enriched in genes involved in RNA metabolism. Accordingly, we find that previously known temperature-sensitive splicing mutants become lethal in the presence of the drug under permissive temperature. Furthermore, in a wild type background, splicing efficiency is decreased and R-loop formation is increased in the presence of formamide. In addition, we have also isolated 35 formamide-sensitive mutants, many of which display remarkable morphology and cell cycle defects potentially unveiling new players in the regulation of these processes. We conclude that formamide preferentially targets RNA related processes in vivo, probably by relaxing RNA secondary structures and/or RNA-protein interactions, and can be used as an effective tool to characterize these processes.
