Identification of bacterial sRNA regulatory targets using ribosome profiling.

Jing Wang,William Rennie,Karine Prévost,Eric Massé,Charles S Carmack,Ye Ding,Chaochun Liu,Marie-Pier Caron,Joseph T Wade

doi:10.1093/nar/gkv1158

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.

Highlights

RNAs represent a major class of regulatory molecule in bacteria
Transient expression of sRNAs has been shown previously to limit indirect regulatory effects (52), and expression of RyhB for
Regulatory targets of RyhB were identified by comparing total RNA levels (RNA-seq component) or ribosome-footprinted RNA levels (Ribo-seq component) for all genes in RyhB-expressing and control cells (Figure 1)

Summary

Introduction

RNAs represent a major class of regulatory molecule in bacteria. ‘Small RNAs’ (sRNAs) are typically non-coding RNAs, 50–150 nt in length (1). Most sRNAs function by interacting with target mRNAs through complementary base pairing, some sRNAs are known to directly interact with proteins. SRNA:mRNA interaction can positively or negatively impact gene expression at the level of translation initiation, mRNA stability or transcription termination (1). The majority of characterized sRNA:mRNA interactions involve the mRNA 5 UTR, and affect mRNA stability and/or translation initiation. Repression of translation typically occurs due to occlusion of the Shine-Dalgarno (S-D) sequence and/or start codon as a result of sRNA binding. Activation of translation typically occurs due to secondary structure alterations around the SD/start codon as a result of sRNA binding to an upstream region on the transcript

Methods

Results

Conclusion