The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

Stella R. Hartono, Amélie Malapert, Pénélope Legros, Pascal Bernard, Frederic Chedin, Vincent Vanoosthuyse

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

Original languageEnglish (US)
Pages (from-to)272-284
Number of pages13
JournalJournal of Molecular Biology
Volume430
Issue number3
DOIs
StatePublished - Feb 2 2018

Fingerprint

Antibody Affinity
Double-Stranded RNA
Schizosaccharomyces
RNA
Ribonuclease H
DNA
Exosome Multienzyme Ribonuclease Complex
Ribonuclease III
Genome
Regulator Genes
Transcriptome
Chromatin
Research Design
Nucleotides
Gene Expression
Antibodies

Keywords

  • DRIPc-seq
  • dsRNA
  • R-loops
  • RNAse H1
  • S9.6

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast. / Hartono, Stella R.; Malapert, Amélie; Legros, Pénélope; Bernard, Pascal; Chedin, Frederic; Vanoosthuyse, Vincent.

In: Journal of Molecular Biology, Vol. 430, No. 3, 02.02.2018, p. 272-284.

Research output: Contribution to journalArticle

Hartono, Stella R. ; Malapert, Amélie ; Legros, Pénélope ; Bernard, Pascal ; Chedin, Frederic ; Vanoosthuyse, Vincent. / The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast. In: Journal of Molecular Biology. 2018 ; Vol. 430, No. 3. pp. 272-284.
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