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

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

Author information

1
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0651, USA.
2
State Key Laboratory of Virology and Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China.
3
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0651, USA; School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430072, China.
4
Department of Pathology, Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093-0651, USA.
5
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0651, USA. Electronic address: xdfu@ucsd.edu.

Abstract

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

PMID:
29104020
PMCID:
PMC5957070
DOI:
10.1016/j.molcel.2017.10.008
[Indexed for MEDLINE]
Free PMC Article

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