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.

DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops.

Unscheduled R loops can be a source of genome instability, a hallmark of cancer cells. Although targeted proteomic approaches and cellular analysis of specific mutants have uncovered factors potentially involved in R-loop homeostasis, we report a more open screening of factors whose depletion causes R loops based on the ability of activation-induced cytidine deaminase (AID) to target R loops. Immunofluorescence analysis of γH2AX caused by small interfering RNAs (siRNAs) covering 3,205 protein-coding genes identifies 59 potential candidates, from which 13 are analyzed further and show a significant increase of R loops. Such candidates are enriched in factors involved in chromatin, transcription, and RNA biogenesis and other processes. A more focused study shows that the DDX47 helicase is an R-loop resolvase, whereas the MeCP2 methyl-CpG-binding protein uncovers a link between DNA methylation and R loops. Thus, our results suggest that a plethora of gene dysfunctions can alter cell physiology via affecting R-loop homeostasis by different mechanisms.

THSC/TREX-2 deficiency causes replication stress and genome instability in Caenorhabditis elegans.

Transcription is an essential process of DNA metabolism, yet it makes DNA more susceptible to DNA damage. THSC/TREX-2 is a conserved eukaryotic protein complex with a key role in mRNP biogenesis and maturation that prevents genome instability. One source of such instability is linked to transcription, as shown in yeast and human cells, but the underlying mechanism and whether this link is universal is still unclear. To obtain further insight into the putative role of the THSC/TREX-2 complex in genome integrity, we have used Caenorhabditis elegans mutants of the thp-1 and dss-1 components of THSC/TREX-2. These mutants show similar defective meiosis, DNA damage accumulation and activation of the DNA damage checkpoint. However, they differ from each other regarding replication defects, as determined by measuring dUTP incorporation in the germline. Interestingly, this specific thp-1 mutant phenotype can be partially rescued by overexpression of RNase H. Furthermore, both mutants show a mild increase in phosphorylation of histone H3 at Ser10 (H3S10P), a mark previously shown to be linked to DNA-RNA hybrid-mediated genome instability. These data support the view that both THSC/TREX-2 factors prevent transcription-associated DNA damage derived from DNA-RNA hybrid accumulation by separate means.

Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability.

R loops are nucleic acid structures comprising an DNA-RNA hybrid and a displaced single-stranded DNA. These structures may occur transiently during transcription, playing essential biological functions. However, persistent R loops may become pathological as they are important drivers of genome instability and have been associated with human diseases. The mitochondrial degradosome is a functionally conserved complex from bacteria to human mitochondria. It is composed of the ATP-dependent RNA and DNA helicase SUV3 and the PNPase ribonuclease, playing a central role in mitochondrial RNA surveillance and degradation. Here we describe a new role for the mitochondrial degradosome in preventing the accumulation of pathological R loops in the mitochondrial DNA, in addition to preventing dsRNA accumulation. Our data indicate that, similar to the molecular mechanisms acting in the nucleus, RNA surveillance mechanisms in the mitochondria are crucial to maintain its genome integrity by counteracting pathological R-loop accumulation.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Diversity, ecology, and evolution in Phycomyces.

The fungal genus Phycomyces (Mucoromycotina, Mucorales) has been revised by examining 96 strains, received from established collections or newly isolated from different environments. Morphology, sexuality, DNA sequences, and population structure clearly identify the genus and set it apart from other Mucorales. The size of the spores, the sexual interactions, the sequences of genes sexM and sexP that determine sexual identity, and the DNA for ribosomal RNAs validate the species Phycomyces blakesleeanus and Phycomyces nitens and the family Phycomycetaceae. Isolates from the same sample of biomass often differ in spore size, sex, DNA sequences, and restriction-length polymorphisms. The widest diversity was found in similar environments in three of the Canary Islands, implying a failure to disperse rapidly over the sea. All strains have apparently functional sexM and sexP genes and all but some strains of P. nitens complete the sexual cycle in the laboratory. The genetic diversity of P. blakesleeanus strains provides evidence for geographical clustering. Various sequence comparisons, including the newly isolated genes sexM and sexP of P. nitens and Blakeslea trispora, clarify phylogenetic relationships in the Mucorales and recommend the sex genes for the study of speciation.