Biology subjects

Richard, S.

Publications and source records attributed to Richard, S..

4 recordsLinked to original sources

bioRxiv2018

Arginine methylation of DDX5 RGG/RG motif by PRMT5 regulates RNA/DNA resolution.

Aberrant transcription-associated RNA:DNA hybrid (R-loop) formation often lead to catastrophic conflicts during replication resulting in DNA double strand breaks and genome instability. To prevent such conflicts, these hybrids require dissolution by helicases and/or RNaseH. Little information is known about how these helicases are regulated. Herein, we identify DDX5, an RGG/RG motif containing DEAD-box family of RNA helicase, as a crucial player in R-loop resolution. We define at the mechanistic level the function of DDX5 in R-loop resolution. In vitro, recombinant DDX5 resolves R-loops in an ATP-dependent manner leading to R-loop degradation by the XRN2 exoribonuclease. DDX5 deficient cells accumulated R-loops at loci known to form R-loops using RNA:DNA immunoprecipitation (DRIP)-qPCR and increased RNaseH sensitive RAD51 foci. PRMT5, an arginine methyltransferase, associated with DDX5 and methylated its RGG/RG motif. This motif was required to associate with XRN2 and resolve cellular R-loops. Furthermore, PRMT5 deficient cells accumulated R-loops, as detected by DRIP-qPCR resulting in increased gH2AX foci. Our findings define a new mechanism by which an RNA helicase, DDX5, is modulated by arginine methylation to resolve R-loops.

molecular biology↗

bioRxiv2018

Site-directed mutagenesis of the 1,3-β glucan synthase catalytic subunit of Pneumocystis jirovecii and susceptibility assays suggest its sensitivity to caspofungin

The echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthetizing 1,3-{beta} glucan, an essential compound of the fungal wall. Studies in rodents showed that caspofungin can treat Pneumocystis infections. However, its efficacy against Pneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of the P. jirovecii Gsc1 subunit, as well as of those of Pneumocystis carinii and Pneumocystis murina infecting respectively rats and mice. In absence of an established in vitro culture method for Pneumocystis species, we used functional complementation of the Saccharomyces cerevisiae gsc1 deletant. In the fungal pathogen Candida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into the Pneumocystis gsc1 genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The minimum inhibitory concentrations of caspofungin determined by E-test(R) and Yeastone(R) for strains complemented with Pneumocystis enzymes (respectively 0.125 and 0.12 microg/ml) were identical to those upon complementation with the enzyme of C. albicans that is sensitive to caspofungin. However, they were lower than the MICs upon complementation with the enzyme of the resistant species Candida parapsilosis (0.19 and 0.25). Sensitivity levels of Gsc1 enzymes of the three Pneumocystis species were similar. Our results suggest that P. jirovecii is sensitive to caspofungin during infections, as P. carinii and P. murina.

microbiology↗

bioRxiv2018

Loss of PRMT5 promotes PDGFRα degradation during oligodendrocyte differentiation and myelination

Platelet derived growth factor receptor (PDGFR) signaling is required for proliferation, commitment and maintenance of oligodendrocyte (OL) precursor cells (OPCs). PDGFR signaling promotes OPC homeostasis and its attenuation signals OPC differentiation and maturation triggering the onset of myelination of the central nervous system (CNS). The initial steps of how PDGFR signaling is attenuated are still poorly understood. Herein we show that decreased Protein Arginine MethylTransferase5 (PRMT5) expression, as occurs during OPC differentiation, is involved in the down-regulation of PDGFR by modulating its cell surface bioavailability leading to its degradation in a Cbldependent manner. Mechanistically, loss of arginine methylation at R554 of the PDGFR intracellular domain reveals a masked Cbl binding site at Y555. Physiologically, depletion of PRMT5 in OPCs results in severe CNS myelination defects. We propose that decreased PRMT5 activity initiates PDGFR degradation to promote OL differentiation. More broadly, inhibition of PRMT5 may be used therapeutically to manipulate PDGFR bioavailability.

molecular biology↗

bioRxiv2017

Mechanisms of surface antigenic variation in the human pathogenic fungus Pneumocystis jirovecii

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii. This uncultivable fungus is an obligate pulmonary pathogen which causes pneumonia in immuno-compromised individuals, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen of a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily which included five families encoding adhesive GPI-anchored glycoproteins, and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination, and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favoured by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists in the continuous production of new subpopulations composed of cells which are antigenically different.

microbiology↗