RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage

Abstract

SummaryR-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease.

Human proteins that interact with RNA/DNA hybrids

“…R-loops immunoprecipitated from HeLa cells with the S9.6 antibody (Cristini et al 2018). Although their study was performed using HeLa cells and our study was carried out with human B-cell extracts, 197 of the proteins identified in their study were also found in our study (Supplemental Table S1).…”

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

“…These data indicate that DDX5/DDX17 is required for transcription termination and transcript release steps of the human HSPA1A gene. We found that DHX9, which was implicated in transcription termination of β‐ and γ‐actin genes (Cristini et al, ), also copurified with hTHO (Supporting information Table S2). However, knockdown of DHX9 did not significantly alter the sizes of the HSP70 mRNA‐containing nuclear foci (Supporting information Figure S1b).…”

“…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.…”

“…As the amount of DDX5 at the HSPA1A pause site was slightly increased by THOC5 knockdown (data not shown), hTHO may not control DDX5/DDX17 recruitment to the heat shock gene but might regulate their activities. For the human β‐ and γ‐actin genes, another abundant DExD/H‐box RNA helicase, DHX9, which was identified as a constituent of the R‐loop interactome, promotes transcriptional termination (Cristini et al, ). Although we identified DHX9 as a hTHO‐interacting factor, we observed, at most, only a marginal effect of knockdown of this RNA helicase on HSP70 mRNA release from the gene locus.…”

“…In addition to the bona fide termination and CPA factors, different RNA/DNA helicases participate in the transcriptional termination and transcript release steps. An RNA/DNA helicase, called senataxin, and a DExD/H‐box RNA helicase, called DHX9, have been shown to resolve RNA/DNA hybrid (R‐loop) structures formed behind elongating RNAP II and thus allow efficient access of XRN2 to the 5′‐end of the cleaved read‐through transcript (Cristini, Groh, Kristiansen, & Gromak, ; Skourti‐Stathaki, Proudfoot, & Gromak, ). Moreover, although the underlying mechanism has not been fully analyzed, p68, a fly ortholog of the human DExD/H‐box RNA helicases DDX5/DDX17, promotes the efficient release of hsp70 mRNA from chromatin (Buszczak & Spradling, ).…”