R-loop mediated genome instability in mRNA cleavage and polyadenylation mutants

Genomic instability via RNA:DNA hybrid mediated R-loops has been observed in various transcription and RNA processing mutants. This study investigates the prevalence of this mechanism across mutants with chromosome instability phenotypes in Saccharomyces cerevisiae. Through direct cytological detection, we identified mutants with elevated RNA:DNA hybrid levels, including mutants of the mRNA cleavage and polyadenylation (mCP) machinery. In parallel, expression of the R-loop-degrading enzyme RNase H was able to suppress a chromosome instability phenotype in some mCP mutants. To elucidate R-loop formation sites across the genome, we are using an RNA:DNA hybrid-specific antibody to perform chromatin immuno-precipitation chip (ChIP-chip) in select mutants with elevated R-loop formation. Furthermore, to verify the conservation of the chromosome instability phenotype in mCP mutants between yeast and humans, we treated human cells with siRNA to knock-down FIP1L1, a conserved mCP component that is a part of an oncogenic fusion in eosinophilic leukemia. Our findings suggest that several RNA processing processes such as mRNA cleavage and polyadenylation when disrupted contribute to increased R-loop formation and genomic instability, which may be relevant to certain human cancers.