title: Identification of bacterial sRNA regulatory targets using ribosome profiling.
abstract: 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.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
authors.forename: William
authors.initials: W
authors.lastname: Rennie
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
authors.forename: Chaochun
authors.initials: C
authors.lastname: Liu
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Carmack
authors.affiliation: RNA Group, Department of Biochemistry, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada.
authors.forename: Karine
authors.initials: K
authors.lastname: Prévost
authors.affiliation: RNA Group, Department of Biochemistry, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada.
authors.forename: Marie-Pier
authors.initials: MP
authors.lastname: Caron
authors.affiliation: RNA Group, Department of Biochemistry, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada.
authors.forename: Eric
authors.initials: E
authors.lastname: Massé
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA Department of Biomedical Sciences, University at Albany, Albany, NY 12201, USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA Department of Biomedical Sciences, University at Albany, Albany, NY 12201, USA joseph.wade@health.ny.gov.
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Wade
mesh_terms: D002374:Catalase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D026901:Membrane Transport Proteins / Q000235:genetics; D016366:Open Reading Frames; D033903:Periplasmic Proteins / Q000235:genetics; D014176:Protein Biosynthesis; D020871:RNA Stability; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033903:Periplasmic Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D058727:RNA, Small Untranslated; C061266:fepB protein, E coli; D002374:Catalase; C051253:katG protein, E coli