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

Identification of bacterial sRNA regulatory targets using ribosome profiling.

Author information

1
Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
2
RNA Group, Department of Biochemistry, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada.
3
Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA Department of Biomedical Sciences, University at Albany, Albany, NY 12201, USA.
4
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.

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.

PMID:
26546513
PMCID:
PMC4666370
DOI:
10.1093/nar/gkv1158
[Indexed for MEDLINE]
Free PMC Article

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