Identification of bacterial sRNA regulatory targets using ribosome profiling.
Identification of bacterial sRNA regulatory targets using ribosome profiling.
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DOI：
10.1093/nar/gkv1158
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发表时间：
2015-12-02
影响因子：
14.9
通讯作者：
Wade JT
中科院分区：
文献类型：
--
作者：
Wang J;Rennie W;Liu C;Carmack CS;Prévost K;Caron MP;Massé E;Ding Y;Wade JT
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.
