R-loop

Abstract: R loops are nucleic acid structures composed of an RNA-DNA hybrid and a displaced single-stranded DNA. Recently, evidence has emerged that R loops occur more often in the genome and have greater physiological relevance, including roles in transcription and chromatin structure, than was previously predicted. Importantly, however, R loops are also a major threat to genome stability. For this reason, several DNA and RNA metabolism factors prevent R-loop formation in cells. Dysfunction of these factors causes R-loop accumulation, which leads to replication stress, genome instability, chromatin alterations or gene silencing, phenomena that are frequently associated with cancer and a number of genetic diseases. We review the current knowledge of the mechanisms controlling R loops and their putative relationship with disease.

Abstract: BRCA2, the breast cancer susceptibility gene factor, interacts with TREX-2, a protein complex involved in the biogenesis and export of messenger ribonucleoprotein, to process DNA–RNA hybrid structures called R-loops that can trigger genome instability; these may be a central cause of the stress occurring in early cancer cells that drives oncogenesis. R-loops — naturally occurring three-stranded nucleic acid structures consisting of an RNA–DNA hybrid and displaced single-stranded DNA — are among the potential inducers of genome instability. This study shows that TREX-2, a complex involved in the biogenesis and export of messenger ribonucleoprotein (mRNP), interacts with the breast cancer susceptibility gene factor BRCA2 to process R-loops. Human cells depleted of BRCA2 accumulate high levels of R-loops. This unexpected interaction between tumour suppressors and R-loops suggests that R-loops may be a major cause of replication stress and tumorigenicity. Genome instability is central to ageing, cancer and other diseases. It is not only proteins involved in DNA replication or the DNA damage response (DDR) that are important for maintaining genome integrity: from yeast to higher eukaryotes, mutations in genes involved in pre-mRNA splicing and in the biogenesis and export of messenger ribonucleoprotein (mRNP) also induce DNA damage and genome instability. This instability is frequently mediated by R-loops formed by DNA–RNA hybrids and a displaced single-stranded DNA1. Here we show that the human TREX-2 complex, which is involved in mRNP biogenesis and export, prevents genome instability as determined by the accumulation of γ-H2AX (Ser-139 phosphorylated histone H2AX) and 53BP1 foci and single-cell electrophoresis in cells depleted of the TREX-2 subunits PCID2, GANP and DSS1. We show that the BRCA2 repair factor, which binds to DSS1, also associates with PCID2 in the cell. The use of an enhanced green fluorescent protein-tagged hybrid-binding domain of RNase H1 and the S9.6 antibody did not detect R-loops in TREX-2-depleted cells, but did detect the accumulation of R-loops in BRCA2-depleted cells. The results indicate that R-loops are frequently formed in cells and that BRCA2 is required for their processing. This link between BRCA2 and RNA-mediated genome instability indicates that R-loops may be a chief source of replication stress and cancer-associated instability.