R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters

Mol Cell. 2017 Nov 16;68(4):745-757.e5. doi: 10.1016/j.molcel.2017.10.008. Epub 2017 Nov 2.


R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

Keywords: Direction of R-loop elongation; Genomic Profiling of R-loops; RNASEH1; Requirement of free RNA end.

MeSH terms

  • DNA / biosynthesis
  • DNA / chemistry*
  • HEK293 Cells
  • Humans
  • K562 Cells
  • Nucleic Acid Heteroduplexes / chemistry*
  • Nucleic Acid Heteroduplexes / metabolism
  • Promoter Regions, Genetic / physiology*
  • RNA / biosynthesis
  • RNA / chemistry*
  • Ribonuclease H / chemistry*
  • Transcription, Genetic*


  • Nucleic Acid Heteroduplexes
  • RNA
  • DNA
  • Ribonuclease H