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

J Mol Biol. 2018 Feb 2;430(3):272-284. doi: 10.1016/j.jmb.2017.12.016. Epub 2017 Dec 28.


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.

Keywords: DRIPc-seq; R-loops; RNAse H1; S9.6; dsRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / chemistry*
  • Immunoprecipitation / methods
  • Nucleic Acid Conformation
  • RNA, Double-Stranded / analysis*
  • RNA, Double-Stranded / genetics
  • RNA, Fungal / analysis*
  • RNA, Fungal / genetics
  • Ribonuclease H / chemistry
  • Schizosaccharomyces / chemistry*
  • Schizosaccharomyces / genetics
  • Sequence Analysis, RNA / methods*
  • Transcriptome


  • Antibodies
  • RNA, Double-Stranded
  • RNA, Fungal
  • Ribonuclease H