Global analysis of RNA cleavage by 5'-hydroxyl RNA sequencing

Nucleic Acids Res. 2015 Sep 30;43(17):e108. doi: 10.1093/nar/gkv536. Epub 2015 May 22.


RNA cleavage by some endoribonucleases and self-cleaving ribozymes produces RNA fragments with 5'-hydroxyl (5'-OH) and 2',3'-cyclic phosphate termini. To identify 5'-OH RNA fragments produced by these cleavage events, we exploited the unique ligation mechanism of Escherichia coli RtcB RNA ligase to attach an oligonucleotide linker to RNAs with 5'-OH termini, followed by steps for library construction and analysis by massively parallel DNA sequencing. We applied the method to RNA from budding yeast and captured known 5'-OH fragments produced by tRNA Splicing Endonuclease (SEN) during processing of intron-containing pre-tRNAs and by Ire1 cleavage of HAC1 mRNA following induction of the unfolded protein response (UPR). We identified numerous novel 5'-OH fragments derived from mRNAs: some 5'-OH mRNA fragments were derived from single, localized cleavages, while others were likely produced by multiple, distributed cleavages. Many 5'-OH fragments derived from mRNAs were produced upstream of codons for highly electrostatic peptides, suggesting that the fragments may be generated by co-translational mRNA decay. Several 5'-OH RNA fragments accumulated during the induction of the UPR, some of which share a common sequence motif that may direct cleavage of these mRNAs. This method enables specific capture of 5'-OH termini and complements existing methods for identifying RNAs with 2',3'-cyclic phosphate termini.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acyl-tRNA Synthetases
  • Endoribonucleases / metabolism*
  • Escherichia coli Proteins
  • High-Throughput Nucleotide Sequencing
  • RNA / chemistry
  • RNA Cleavage*
  • RNA Stability
  • RNA, Messenger / metabolism
  • Saccharomyces cerevisiae / genetics
  • Sequence Analysis, RNA / methods*
  • Unfolded Protein Response


  • Escherichia coli Proteins
  • RNA, Messenger
  • RNA
  • Endoribonucleases
  • Amino Acyl-tRNA Synthetases
  • RtcB protein, E coli