An ATR‐dependent function for the Ddx19 RNA helicase in nuclear R‐loop metabolism

Abstract

Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells.

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells

“…Initial experiments were performed with purified bacterially-expressed DDX19B (denoted DDX19B*) (Figure S4); however, the bacterially-expressed DDX19B* with or without hgle1B-CTD had ~8.3 fold lower enzymatic activity compared to that of S. cerevisiae Dbp5, gle1-CTD and IP 6 (Figure S4). We then tested His-tagged DDX19B (H 6 -DDX19B) purified from a Baculoviral expression system in insect cells based on reported successful DDX19B in vitro protein interaction studies and reports that DDX19B is post-translationally modified in mammalian cells 40,41 (Figure 6A), The H 6 -DDX19B purified from insect cells displayed significant ATPase activity which was stimulated ~4-fold by hgle1B-CTD in the presence of IP 6 (Figure 6B; lane 2, 6, 7). Strikingly, titration of hnup42-CTD resulted in dose-dependent enhancement of the hGle1B-stimulated H 6 -DDX19B ATPase activity in the absence of IP 6 (Figure 6B, lanes 8–10).…”

“…A recent study reported roles for DDX19B phosphorylation in altering its nuclear activity during the DNA damage response 40 . We show here that DDX19B* purified from bacteria is not activated by hgle1-CTD in the presence of IP 6 or hnup42-CTD, and demonstrates relatively low enzymatic activity (Figure S4).…”

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

“…To examine the role of the DDX5 in R‐loop resolution at precise genomic loci, we used the RNA:DNA immunoprecipitation (DRIP) method. By qPCR analysis, we quantified R‐loop accumulation at four selected loci, EGR1, MALAT1, HIST1H2BG, and RPPH1 , previously known to have a propensity to form R‐loops (Yang et al , ; García‐Rubio et al , ; Hodroj et al , ). The R‐loops for EGR1 and MALAT1 reside within the gene body, while the R‐loops for HIST1H2BG and RPPH1 encompass the transcription termination region, as determined previously and presented in the R‐loop database (Fig D) (Wongsurawat et al , ).…”

“…Senataxin is reported to function with the 5′–3′ exonuclease XRN2 to resolve a subset of R‐loops at transcription termination sites of actively transcribed genes (Skourti‐Stathaki et al , ; Morales et al , ; Aymard et al , ). The DNA helicase RECQ5 and RNA helicases DDX1 (Li et al , , ; Ribeiro de Almeida et al , ), DDX19 (Hodroj et al , ), DDX21 (Song et al , ), DDX23 (Sridhara et al , ), and DHX9 (Cristini et al , ) were also found to be functionally involved in suppression of R‐loops. Topoisomerase I removes the negative supercoils behind RNA polymerases to prevent annealing of the nascent RNA with the DNA template and suppresses R‐loop formation (Tuduri et al , ).…”

Human THO coordinates transcription termination and subsequent transcript release from the HSP70 locus

“…Aberrant R‐loops formed in the absence of THO interrupt DNA replication and induce genome instability (Gómez‐González et al, ; Santos‐Pereira & Aguilera, ). It has also been shown that various DNA and RNA helicases as well as XRN2 prevent the formation of transcriptional R‐loops (Boulé & Zakian, ; Cristini et al, ; Hodroj et al, ; Kim, Choe, & Seo, ; Morales et al, ). To test the possibility that aberrant R‐loop formation contributes to transcript accumulation of the human HSPA1A gene, we over‐expressed RNase H in THOC5‐ and DDX5/DDX17‐depleted cells, and HSP70 mRNA localization was analyzed by in situ hybridization.…”