Activation of gene expression by small RNA

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Small regulatory RNAs (sRNAs) commonly act to downregulate gene expression. In bacteria, however, sRNAs have also been shown to activate genes by a variety of direct or indirect mechanisms. Several sRNAs (DsrA, GlmZ, RNAIII, RprA, RyhB, and Qrr) act as direct translational activators by an ‘anti-antisense mechanism’ in the 5′ mRNA region to liberate a sequestered ribosome binding site, while pairing of GadY sRNA to the 3′-end alters processing and increases stability of its target mRNA. Indirect activation includes cases of RNA mimicry in which degradation of the activating GlmZ sRNA is suppressed by the highly similar GlmY sRNA, or where a pseudo-target mRNA traps MicM sRNA to derepress porin synthesis.

Introduction

The two most prominent classes of regulatory small RNAs to date, the ∼22 nt microRNAs of eukaryotes [1] and the 50–400 nt sRNAs of bacteria [2], both act to modulate gene expression at the post-transcriptional level. The microRNAs in general, and most of the bacterial sRNAs, exert their regulatory function by short base-pairing to target mRNAs. The interactions of microRNAs with the 3′-untranslated region (UTR) or coding sequence (CDS) of targets almost exclusively results in the repression of the encoded gene, by a variety of mechanisms including translational control, induced mRNA cleavage and deadenylation [3]. Few cases of microRNA-mediated activation have been reported [1].

Likewise, the common base-pairing of bacterial sRNAs to the 5′ mRNA region most often represses the target [2]. Typically, sRNA pairing masks the ribosome binding site (RBS) of the target, thus inhibiting 30S ribosomal subunit association and translational initiation. As a consequence, the untranslated target mRNA is destabilized, usually by the action of RNase E [4, 5] or RNase III [6, 7], although cases of translational repression without mRNA stability changes were also reported (e.g. [8]). Downregulation by sRNAs is not limited to RBS targeting: several sRNAs inhibit translation in the upstream 5′-UTR [9, 10, 11], or promote target decay without translational repression in the CDS [12] or intergenic space of polycistronic mRNA [13].

At least in Gram-negative bacteria, a considerable number of base-pairing sRNAs require the RNA chaperone Hfq [2] for both intracellular stability and productive target pairing [14]. In fact, most of the sRNAs described below are Hfq-dependent. Deletion of the hfq gene causes diverse phenotypes impairing both general physiology and virulence of a wide range of bacteria, and deregulates up to one-fifth of all genes in enteric bacteria such as Escherichia coli and Salmonella [15, 16]. Not only upregulation of distinct genes (indicating loss of repression) but also downregulation is observed in hfq deletion mutants; as far as post-transcriptional regulation is concerned, such downregulation may in part reflect a loss-of-function of activating sRNAs.

The present review covers the two mechanisms known to date by which direct base-pairing of sRNAs activates a target mRNA, that is the ‘anti-antisense mechanism’ and the 3′-processing-mediated transcript stabilization (Figure 1A,B). In addition to direct activation, we will describe the cases of the GlmYZ and MicM sRNAs in which RNA mimicry leads to target activation through the backdoor (Figure 1C,D).

Bacterial messengers are not the only targets, and a number of sRNAs are known to bind cellular proteins to modulate their activity. There is currently no well-established molecular mechanism for direct stimulation of protein activity by sRNA. However, both the antagonistic binding of the CsrB-like sRNAs to CsrA/RsmA proteins, and the association of 6S RNA with RNA polymerase (RNAP), can eventually lead to increased gene expression. These regulations and their physiological consequences have been recently reviewed [17, 18, 19].

Section snippets

Direct translational activation of mRNA: the anti-antisense mechanism

Translation is initiated by sequence-specific anchoring of 30S ribosomes at the RBS as defined by the Shine–Dalgarno (SD) sequence and the AUG start codon. If any part of the extended RBS region from approximately −35 relative to the translational start down to the fifth codon is sequestered by a stable RNA structure, the rate-limiting initiation step is inefficient, resulting in low protein synthesis [20]. Hairpins that sequester the RBS are not uncommon in bacterial mRNAs, and often underlie

Comparison of translational activation and repression

The known activating and inhibitory sRNAs act by similarly short and imperfect RNA interactions; the Qrr sRNAs even use the very same region to activate and repress mRNAs [49]. Nonetheless, the known activation sites are ≥22 nt upstream of the target AUG (Table 1), whereas repression commonly takes place proximal (<20 nt) to AUG [2, 50] and also in the CDS [12, 20, 51]. However, since sRNAs can repress translation 100 nt upstream of AUG [9], the sRNA binding site position by itself cannot safely

3′-End-mediated mRNA stabilization

Antisense RNAs that are encoded in cis and in opposite orientation to their target mRNAs constitute a large class of bacterial regulators. Originally identified on bacterial mobile elements [53], and now increasingly so in bacterial chromosomes as well [54], the cis-encoded antisense RNAs have consistently been reported to repress mRNAs, with one exception: GadY of E. coli [55]. This sRNA is encoded on the opposite strand in the IGR of gadX and gadW, the two downstream ORFs in the gadAXW acid

Indirect activation by saving the direct activator: the GlmYZ sRNAs

We outlined above how glmS mRNA is activated by GlmZ through anti-antisense pairing. However, the first sRNA found to stimulate GlmS synthesis was GlmY [59], and overexpression of the two sRNAs invariably causes GlmS accumulation [34]. GlmY and GlmZ share substantial sequence and structure homology; crucially, however, GlmY lacks the complementary region to glmS mRNA [34, 60], and unlike GlmZ fails to promote glmS translation in vitro [34]. How does then GlmY stimulate GlmS synthesis?

In

An mRNA trap removes an inhibitory sRNA to activate another mRNA

While the GlmYZ case has been suggestive of RNA mimicry, direct RNA competition that ultimately increased mRNA expression has recently been identified in the regulation of YbfM, a chitosugar-specific porin of E. coli and Salmonella [62, 63] (Figure 1D). The ybfM mRNA is strongly repressed under standard growth conditions by RBS pairing of the constitutively expressed, trans-encoded MicM sRNA [64]. Unlike in other Hfq-dependent repressions [5], the MicM–ybfM pairing does not entail coupled

How many activator RNAs are there?

It is now well established that conserved sRNAs often regulate multiple target mRNAs [2, 52]. We expect that, similar to DsrA, RNAIII, and RyhB, sRNAs with multiple targets might often include an activated mRNA in their regulon. Biocomputational algorithms such as TargetRNA [66] or RNAhybrid [67] can predict target interactions yet do not take possible activation into account. Candidates for activation through anti-antisense pairing might be identified in the available catalogs of

Acknowledgements

We thank the members of our laboratory for comments on the manuscript, and colleagues for sharing unpublished results. Work in the Vogel lab is supported by the Deutsche Forschungsgemeinschaft (DFG) Priority Program SPP1258 Sensory and Regulatory RNAs in Prokaryotes. KSF is supported by the International Max Planck Research School for Infectious Diseases and Immunology Program.

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