Identification of bacterial sRNA regulatory targets using ribosome profiling

Nucleic Acids Res. 2015 Dec 2;43(21):10308-20. doi: 10.1093/nar/gkv1158. Epub 2015 Nov 5.


Bacteria express large numbers of non-coding, regulatory RNAs known as 'small RNAs' (sRNAs). sRNAs typically regulate expression of multiple target messenger RNAs (mRNAs) through base-pairing interactions. sRNA:mRNA base-pairing often results in altered mRNA stability and/or altered translation initiation. Computational identification of sRNA targets is challenging due to the requirement for only short regions of base-pairing that can accommodate mismatches. Experimental approaches have been applied to identify sRNA targets on a genomic scale, but these focus only on those targets regulated at the level of mRNA stability. Here, we utilize ribosome profiling (Ribo-seq) to experimentally identify regulatory targets of the Escherichia coli sRNA RyhB. We not only validate a majority of known RyhB targets using the Ribo-seq approach, but also discover many novel ones. We further confirm regulation of a selection of known and novel targets using targeted reporter assays. By mutating nucleotides in the mRNA of a newly discovered target, we demonstrate direct regulation of this target by RyhB. Moreover, we show that Ribo-seq distinguishes between mRNAs regulated at the level of RNA stability and those regulated at the level of translation. Thus, Ribo-seq represents a powerful approach for genome-scale identification of sRNA targets.

Publication types

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

MeSH terms

  • Catalase / genetics
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Gene Expression Regulation, Bacterial*
  • Membrane Transport Proteins / genetics
  • Open Reading Frames
  • Periplasmic Proteins / genetics
  • Protein Biosynthesis*
  • RNA Stability
  • RNA, Bacterial / metabolism*
  • RNA, Messenger / chemistry
  • RNA, Messenger / metabolism
  • RNA, Small Untranslated / metabolism*
  • Ribosomes / metabolism
  • Sequence Analysis, RNA / methods*


  • Escherichia coli Proteins
  • Membrane Transport Proteins
  • Periplasmic Proteins
  • RNA, Bacterial
  • RNA, Messenger
  • RNA, Small Untranslated
  • fepB protein, E coli
  • Catalase
  • katG protein, E coli