Multiple target regulation by small noncoding RNAs rewires gene expression at the post-transcriptional level

Abstract

Small noncoding RNAs (sRNAs), often in conjunction with Hfq protein, have increasingly been shown to regulate multiple rather than individual mRNAs, thereby reprogramming gene expression at the post-transcriptional level. This review summarizes how and when several such regulators (CyaR, DsrA, GcvB, OmrAB, RNAIII, RybB, RyhB) act upon multiple targets.

Introduction

Bacterial adaptation to changing environmental conditions requires dynamic gene expression changes within specific regulatory circuits. Transcriptional networks controlled by DNA binding proteins orchestrate the expression of physiologically relevant genes in response to extra- or intracellular stimuli. Transcriptional repression or activation was long considered the only relevant way to switch bacterial genes on and off. However, post-transcriptional control at the mRNA level is now generally considered to play a pivotal role in bacterial gene expression as well.

Small noncoding RNAs (sRNAs) are the most abundant class of post-transcriptional regulators in bacteria. Following the original discovery of MicF sRNA as a trans-encoded regulator of Escherichia coli ompF mRNA [57] and the advent of systematic genome-wide sRNA searches in the year 2001 (reviewed in [96]), the total number of known E. coli and Salmonella sRNAs has grown to well over a hundred. In addition, hundreds, if not thousands, of candidate sRNAs have been predicted in a wide range of bacteria (reviewed in [48]).

The prototypic sRNAs of enterobacterial species come in a typical size range of ∼50 to 200 nucleotides, are usually encoded by free-standing genes and are often expressed under specific growth, stress or virulence conditions. Whilst some sRNAs act to modulate the activity of proteins [6], [15], [98], the majority are believed to modulate gene expression by direct base-pairing with mRNAs [97]. Regulation is predominantly negative and may primarily occur at the level of translation initiation [2], yet sRNAs can also activate the translation of a target mRNA and regulate by altering messenger stability.

In many cases, the RNA chaperone Hfq is required for productive target pairing, presumably by actively remodeling the interacting RNAs to melt inhibitory secondary structures or by serving passively as a platform to bring together sRNAs and target mRNAs, effectively increasing local RNA concentrations [2], [12], [92]. The relevance of Hfq-mediated regulation is well reflected by the pleiotropic phenotypes reported for hfq-deficient cells in various bacteria [22], [76], [80], [86], and by the complexity of the regulatory circuits, e.g., virulence gene expression and quorum sensing, in which Hfq and sRNAs were discovered to function [46], [85].

Early studies often focused on regulation of a single target for a given sRNA; in fact, ompF mRNA has remained the only investigated target of MicF sRNA in 25 years. In contrast, OxyS sRNA was early on observed to alter the steady-state levels of >40 abundant E. coli proteins [3]. Likewise, pioneering work on E. coli DsrA revealed that such regulation can be direct, i.e. this sRNA directly interacted with more than one mRNA [44], [49].

There has since been increasing evidence that sRNAs may indeed more often than not regulate multiple targets.

First, many structural and genetic studies have led us to fully appreciate that sRNA-target interactions are usually short (≤25 bp) and imperfect, with core interactions as short as six non-redundant base pairs in the E. coli SgrS-ptsG interaction [40]. Given these short interactions, any sRNA may have enough sequence space for multiple binding partners. Second, sRNA pairing to the Shine–Dalgarno (SD) or AUG start codon sequence was long considered a hallmark of productive target repression. However, several groups have recently reported regulation outside this narrow and low sequence complexity region [11], [18], [73], [93]; such studies have expanded the sequence space for productive targeting on the mRNA side. Third, global approaches using co-immunoprecipitation coupled to microarray [101] or deep sequencing analysis [75] have identified numbers of Hfq-associated mRNAs that surpass those of known sRNAs, e.g., ≥700 mRNAs versus ∼100 sRNAs in Salmonella ([75], [74] and unpublished results). Thus, there seem to be more potential targets than regulators. Fourth, new microarray-based experimental approaches using sRNA pulse expression [55], [65], [84] or sRNA-mediated pulldown of interacting mRNAs [23] have predicted with high confidence that diverse sRNAs directly regulate more than one mRNA.

In this review, we will focus on several well-investigated sRNAs (Fig. 1) that regulate gene expression by the interaction with multiple mRNAs. For mechanisms of global gene regulation by sRNAs through the interaction with proteins, e.g., by the 6S or CsrB-like RNAs, the reader is referred to excellent reviews by others [6], [42], [98].

We have selected for review DsrA, the first intensely studied multiple target regulator; GcvB, which recognizes mRNAs of many ABC transport systems; RyhB having an extensive role under iron-replete conditions; four sRNAs (CyaR, OmrAB, RybB) that modulate outer membrane properties; and RNAIII, a regulator of sundry virulence factor mRNAs.