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

Oscar D Villarreal,Jean-Yves Masson,Zhenbao Yu,Sofiane Y Mersaoui,Stéphane Richard

doi:10.26508/lsa.202000762

Abstract

DDX5, XRN2, and PRMT5 have been shown to resolve DNA/RNA hybrids (R-loops) at RNA polymerase II transcription termination sites at few genomic loci. Herein, we perform genome-wide R-loop mapping using classical DNA/RNA immunoprecipitation and high-throughput sequencing (DRIP-seq) of loci regulated by DDX5, XRN2, and PRMT5. We observed hundreds to thousands of R-loop gains and losses at transcribed loci in DDX5-, XRN2-, and PRMT5-deficient U2OS cells. R-loop gains were characteristic of highly transcribed genes located at gene-rich regions, whereas R-loop losses were observed in low-density gene areas. DDX5, XRN2, and PRMT5 shared many R-loop gain loci at transcription termination sites, consistent with their coordinated role in RNA polymerase II transcription termination. DDX5-depleted cells had unique R-loop gain peaks near the transcription start site that did not overlap with those of siXRN2 and siPRMT5 cells, suggesting a role for DDX5 in transcription initiation independent of XRN2 and PRMT5. Moreover, we observed that the accumulated R-loops at certain loci in siDDX5, siXRN2, and siPRMT5 cells near the transcription start site of genes led to antisense intergenic transcription. Our findings define unique and shared roles of DDX5, XRN2, and PRMT5 in DNA/RNA hybrid regulation.

Highlights

R-loops are three-stranded structures consisting of a DNA/RNA hybrid and the displaced strand of single-stranded DNA
We showed that DDX5, XRN2, and PRMT5-deficient cells accumulate R-loops at the termination sites (TTS) of specific loci in U2OS cells (Mersaoui et al, 2019)
We performed DNA/RNA immunoprecipitation (IP) and high-throughput sequencing (DRIP-seq) to identify the genome-wide R-loops regulated by DDX5, XRN2, and PRMT5

Summary

Introduction

R-loops are three-stranded structures consisting of a DNA/RNA hybrid and the displaced strand of single-stranded DNA. R-loops are typically, but not exclusively, formed co-transcriptionally where there is reannealing of the nascent transcript to its complementary DNA template with an estimated frequency of 5% depending on the locus and its sequence, transcription levels, and overall gene length (Sanz et al, 2016; Stork et al, 2016; Wahba et al, 2016). These R-loops are mainly associated with accessible chromatin at the transcription start sites (TSS) of gene promoters and at the transcription termination sites (TTS) (Manzo et al, 2018). R-loops are necessary for immunoglobulin class switch recombination targeting activation-induced cytidine deaminase (AID) to the IgH S-regions (Yu et al, 2003; Ribeiro de Almeida et al, 2018)

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