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Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase H Targets in tRNA Genes, Retrotransposons and Mitochondria


R-loops (RNA-DNA hybrids) are potentially deleterious for gene expression and genome stability, but can be beneficial, for example, during immunoglobulin gene class-switch recombination. Here we made use of antibody S9.6, with specificity for RNA-DNA duplexes independently of their sequence. The genome-wide distribution of R-loops in wild-type yeast showed association with the highly transcribed ribosomal DNA, and protein-coding genes, particularly the second exon of spliced genes. On RNA polymerase III loci such as the highly transcribed transfer RNA genes (tRNAs), R-loop accumulation was strongly detected in the absence of both ribonucleases H1 and H2 (RNase H1 and H2), indicating that R-loops are inherently formed but rapidly cleared by RNase H. Importantly, stable R-loops lead to reduced synthesis of tRNA precursors in mutants lacking RNase H and DNA topoisomerase activities. RNA-DNA hybrids associated with TY1 cDNA retrotransposition intermediates were elevated in the absence of RNase H, and this was accompanied by increased retrotransposition, in particular to 5′-flanking regions of tRNAs. Our findings show that RNase H participates in silencing of TY1 life cycle. Surprisingly, R-loops associated with mitochondrial transcription units were suppressed specifically by RNase H1. These findings have potentially important implications for understanding human diseases caused by mutations in RNase H.


Vyšlo v časopise: Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase H Targets in tRNA Genes, Retrotransposons and Mitochondria. PLoS Genet 10(10): e32767. doi:10.1371/journal.pgen.1004716
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004716

Souhrn

R-loops (RNA-DNA hybrids) are potentially deleterious for gene expression and genome stability, but can be beneficial, for example, during immunoglobulin gene class-switch recombination. Here we made use of antibody S9.6, with specificity for RNA-DNA duplexes independently of their sequence. The genome-wide distribution of R-loops in wild-type yeast showed association with the highly transcribed ribosomal DNA, and protein-coding genes, particularly the second exon of spliced genes. On RNA polymerase III loci such as the highly transcribed transfer RNA genes (tRNAs), R-loop accumulation was strongly detected in the absence of both ribonucleases H1 and H2 (RNase H1 and H2), indicating that R-loops are inherently formed but rapidly cleared by RNase H. Importantly, stable R-loops lead to reduced synthesis of tRNA precursors in mutants lacking RNase H and DNA topoisomerase activities. RNA-DNA hybrids associated with TY1 cDNA retrotransposition intermediates were elevated in the absence of RNase H, and this was accompanied by increased retrotransposition, in particular to 5′-flanking regions of tRNAs. Our findings show that RNase H participates in silencing of TY1 life cycle. Surprisingly, R-loops associated with mitochondrial transcription units were suppressed specifically by RNase H1. These findings have potentially important implications for understanding human diseases caused by mutations in RNase H.


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