Abstract
DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013.
Highlights
Elevated DNA:RNA hybrid formation due to defects in RNA processing pathways leads to genome instability and replication stress across species [1,2,3,4,5,6,7]
We show that overexpression of RNase H, which degrades the RNA in hybrids, significantly affects the expression of genes associated with antisense transcripts
DNA:RNA immunoprecipitation (DRIP) coupled with deep sequencing in human cells has demonstrated the prevalence of R loops at CpG island promoters with high GC skew [26]
Summary
Elevated DNA:RNA hybrid formation due to defects in RNA processing pathways leads to genome instability and replication stress across species [1,2,3,4,5,6,7]. R loops threaten genome stability and often form under abnormal conditions where nascent mRNA is improperly processed or RNA half-life is increased, resulting in RNA that can hybridize with template DNA, displacing the nontranscribed DNA strand [8]. A recent study found that hybrid formation can occur in trans via Rad51-mediated DNA-RNA strand exchange [9]. The second, more widespread mechanism, identified in Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans and human cells, involves the R loops and associated stalled transcription complexes, which block DNA replication fork progression [3,4,8,10,11]. Mutations in RNA splicing/ processing factors are frequently found in human cancer, heritable diseases like Aicardi-Goutieres syndrome, and a degenerative ataxia associated with Senataxin mutations [13,14,15,16,17]
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