RNA:DNA hybrid structures known as R loops were thought to be rare byproducts of transcription. In the last decade, however, accumulating evidence has pointed to a new view in which R loops form more frequently, impacting transcription and threatening genome integrity as a source of chromosome fragility and a potential cause of disease. Not surprisingly, cells have evolved mechanisms to prevent cotranscriptional R loop formation. Here we discuss the factors and cellular processes that control R loop formation and the mechanisms by which R loops may influence gene expression and the integrity of the genome.

No abstract

Genetic instability, a phenomenon relevant for developmentally regulated processes, cancer, and inherited disorders, can be induced by transcription. However, the mechanisms of transcription-associated genetic instability are not yet understood. Analysis of S. cerevisiae mutants of THO/TREX, a conserved eukaryotic protein complex functioning at the interface of transcription and mRNA metabolism, has provided evidence that transcription elongation impairment can cause hyperrecombination. Here we show, using hpr1Delta mutants, that the nascent mRNA can diminish transcription elongation efficiency and promote recombination. If during transcription the nascent mRNA is self-cleaved by a hammerhead ribozyme, the transcription-defect and hyperrecombination phenotypes of hpr1Delta cells are suppressed. Abolishment of hyperrecombination by overexpression of RNase H1 and molecular detection of DNA:RNA hybrids indicate that these are formed cotranscriptionally in hpr1Delta cells. These data support a model to explain the connection between recombination, transcription, and mRNA metabolism and provide a new perspective to understanding transcription-associated recombination.

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.

Genomic instability in the form of mutations and chromosome rearrangements is usually associated with pathological disorders, and yet it is also crucial for evolution. Two types of elements have a key role in instability leading to rearrangements: those that act in trans to prevent instability--among them are replication, repair and S-phase checkpoint factors--and those that act in cis--chromosomal hotspots of instability such as fragile sites and highly transcribed DNA sequences. Taking these elements as a guide, we review the causes and consequences of instability with the aim of providing a mechanistic perspective on the origin of genomic instability.

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 DNA. 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.

Summary R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability, however the mechanisms underlying R-loop induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively. These factors couple the machineries that function in splicing and export of mRNA. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex, which functions in transcription in yeast. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)(+) RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX ('transcription/export') complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.