DICER ribonuclease removes harmful R-loops.

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

UAP56/DDX39B is a major cotranscriptional RNA-DNA helicase that unwinds harmful R loops genome-wide.

Nonscheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R-loop accumulation. One mechanism relies on the conserved THO complex in association with cotranscriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R-loop removal. We show that UAP56 depletion causes R-loop accumulation, R-loop-mediated genome instability, and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and show that its overexpression suppresses R loops and genome instability induced by depleting five different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful cotranscriptional RNA structures that otherwise would block transcription and replication.

The THO Complex as a Paradigm for the Prevention of Cotranscriptional R-Loops.

Different proteins associate with the nascent RNA and the RNA polymerase (RNAP) to catalyze the transcription cycle and RNA export. If these processes are not properly controlled, the nascent RNA can thread back and hybridize to the DNA template forming R-loops capable of stalling replication, leading to DNA breaks. Given the transcriptional promiscuity of the genome, which leads to large amounts of RNAs from mRNAs to different types of ncRNAs, these can become a major threat to genome integrity if they form R-loops. Consequently, cells have evolved nuclear factors to prevent this phenomenon that includes THO, a conserved eukaryotic complex acting in transcription elongation and RNA processing and export that upon inactivation causes genome instability linked to R-loop accumulation. We revise and discuss here the biological relevance of THO and a number of RNA helicases, including the THO partner UAP56/DDX39B, as a paradigm of the cellular mechanisms of cotranscriptional R-loop prevention.

Human THO-Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability.

R-loops, formed by co-transcriptional DNA-RNA hybrids and a displaced DNA single strand (ssDNA), fulfill certain positive regulatory roles but are also a source of genomic instability. One key cellular mechanism to prevent R-loop accumulation centers on the conserved THO/TREX complex, an RNA-binding factor involved in transcription elongation and RNA export that contributes to messenger ribonucleoprotein (mRNP) assembly, but whose precise function is still unclear. To understand how THO restrains harmful R-loops, we searched for new THO-interacting factors. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R-loops, DNA damage, and replication impairment. Functional analyses show that histone hypo-acetylation prevents accumulation of harmful R-loops and RNA-mediated genomic instability. Diminished histone deacetylase activity in THO- and Sin3A-depleted cell lines correlates with increased R-loop formation, genomic instability, and replication fork stalling. Our study thus uncovers physical and functional crosstalk between RNA-binding factors and chromatin modifiers with a major role in preventing R-loop formation and RNA-mediated genome instability.

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops.

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.

Coenzyme q10 therapy.

For a number of years, coenzyme Q10 (CoQ10) was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in blood plasma, and extensively investigated its antioxidant role. These 2 functions constitute the basis for supporting the clinical use of CoQ10. Also, at the inner mitochondrial membrane level, CoQ10 is recognized as an obligatory cofactor for the function of uncoupling proteins and a modulator of the mitochondrial transition pore. Furthermore, recent data indicate that CoQ10 affects the expression of genes involved in human cell signaling, metabolism and transport, and some of the effects of CoQ10 supplementation may be due to this property. CoQ10 deficiencies are due to autosomal recessive mutations, mitochondrial diseases, aging-related oxidative stress and carcinogenesis processes, and also statin treatment. Many neurodegenerative disorders, diabetes, cancer, and muscular and cardiovascular diseases have been associated with low CoQ10 levels as well as different ataxias and encephalomyopathies. CoQ10 treatment does not cause serious adverse effects in humans and new formulations have been developed that increase CoQ10 absorption and tissue distribution. Oral administration of CoQ10 is a frequent antioxidant strategy in many diseases that may provide a significant symptomatic benefit.