Arginine methylation of the DDX 5 helicase RGG / RG motif by PRMT 5 regulates resolution of RNA:DNA hybrids

Abstract

Aberrant transcription‐associated RNA : DNA hybrid (R‐loop) formation often causes catastrophic conflicts during replication, resulting in DNA double‐strand breaks and genomic instability. Preventing such conflicts requires hybrid dissolution by helicases and/or RN ase H. Little is known about how such helicases are regulated. Herein, we identify DDX 5, an RGG / RG motif‐containing DEAD ‐box family RNA helicase, as crucial player in R‐loop resolution. In vitro , recombinant DDX 5 resolves R‐loops in an ATP ‐dependent manner, leading to R‐loop degradation by the XRN 2 exoribonuclease. DDX 5‐deficient cells accumulate R‐loops at loci with propensity to form such structures based on RNA : DNA immunoprecipitation ( DRIP )‐ qPCR , causing spontaneous DNA double‐strand breaks and hypersensitivity to replication stress. DDX 5 associates with XRN 2 and resolves R‐loops at transcriptional termination regions downstream of poly(A) sites, to facilitate RNA polymerase II release associated with transcriptional termination. Protein arginine methyltransferase 5 ( PRMT 5) binds and methylates DDX 5 at its RGG / RG motif. This motif is required for DDX 5 interaction with XRN 2 and repression of cellular R‐loops, but not essential for DDX 5 helicase enzymatic activity. PRMT 5‐deficient cells accumulate R‐loops, resulting in increased formation of γH2 AX foci. Our findings exemplify a mechanism by which an RNA helicase is modulated by arginine methylation to resolve R‐loops, and its potential role in regulating transcription.

The balancing act of R-loop biology: The good, the bad, and the ugly

“…DDX5, a DEAD-box RNA helicase, has been reported to have a DNA:RNA hybrid unwinding activity (50,51). A recent study using human cell lines found that DDX5 interacts with XRN2 at transcriptional termination regions through its RGG/RG motif to resolve R-loops (51).…”

“…DDX5, a DEAD-box RNA helicase, has been reported to have a DNA:RNA hybrid unwinding activity (50,51). A recent study using human cell lines found that DDX5 interacts with XRN2 at transcriptional termination regions through its RGG/RG motif to resolve R-loops (51). Although it is not essential for the DDX5 helicase activity in vitro, methylation of the RGG/RG motif of DDX5 by protein arginine methyltransferase 5 (PRMT5) was required for DDX5 to interact with XRN2 and promote R-loop resolution.…”

Genome-wide R-loop analysis defines unique roles for DDX5, XRN2, and PRMT5 in DNA/RNA hybrid resolution

“…In addition, when formed, R-loops can be removed by cellular RNA nucleases, including RNase H1 and RNase H2, which specifically cleave the RNA moiety of the R-loops (Wahba et al, 2011) and the 59-39 exoribonuclease 2 (XRN2) which degrades nascent RNA downstream the 39-terminal cleavage site to facilitate transcription termination. XRN2 physically associates with helicase such as Senataxin (Skourti-Stathaki et al, 2011) and DDX5 (Mersaoui et al, 2019). The RNA helicases resolve the DNA/RNA hybrids for subsequent degradation by XRN2 (Skourti-Stathaki et al, 2011;Mersaoui et al, 2019).…”

“…XRN2 physically associates with helicase such as Senataxin (Skourti-Stathaki et al, 2011) and DDX5 (Mersaoui et al, 2019). The RNA helicases resolve the DNA/RNA hybrids for subsequent degradation by XRN2 (Skourti-Stathaki et al, 2011;Mersaoui et al, 2019). Deficiency of XRN2 expression causes R-loop accumulation and DNA damage (Morales et al, 2016;Mersaoui et al, 2019).…”

Mechanistic Dissection of RNA-Binding Proteins in Regulated Gene Expression at Chromatin Levels

“…2C; Chen Fig. 2C; Argaud et al 2019;Mersaoui et al 2019). Pre-mRNA splicing and RNA export factors appear to help pull nascent RNAs out from R-loops to facilitate RNA processing and transport, thus preventing R-loop accumulation ( Fig.…”