Mechanism of RNA silencing by Hfq-binding small RNAs
Introduction
Regulation of gene expression by small RNAs (sRNAs) has been known for many years in Escherichia coli since the serendipitous discovery of MicF, which is involved in the downregulation of expression of ompF, encoding a major outer membrane porin, OmpF [1]. The characterization of several additional sRNAs such as DsrA and OxyS, that were also found fortuitously [2, 3] (along with the identification of numerous sRNAs by systematic searches [4, 5, 6, 7, 8, 9]), have shown that sRNAs are widely involved in the regulation of gene expression, primarily at post-transcriptional levels. Although the biological functions of many of the newly identified sRNAs remain to be elucidated, it is becoming clear that a major class of E. coli sRNAs bind to an RNA chaperone, Hfq [10], and act by imperfect base-pairing to regulate, in most cases negatively, the translation and stability of target mRNAs under specific stress conditions. The physiological roles and the mechanisms of action of these sRNAs have been extensively reviewed in the last few years [11, 12, 13, 14, 15, 16, 17, 18]. What has been learned about the mechanisms of action of Hfq-binding sRNAs, with a focus on newly emerging features that have been uncovered by the studies on two sRNAs, SgrS [19•] and RyhB [20], that downregulate target mRNAs under glucose phosphate stress and iron-depletion conditions, respectively, is discussed here.
Section snippets
RNAse E-dependent coupled degradation of target mRNAs and sRNAs
RyhB RNA, one of the Hfq-binding sRNAs identified by systematic searches [4, 6], downregulates several genes for iron-binding proteins including sodB, which encodes superoxide dismutase, in response to iron limitation [20, 21]. RyhB causes a rapid degradation of target mRNAs in an RNAse E-dependent manner [22•]. The rapid degradation of target mRNAs was thought to be an additional mode of sRNA function. It should be noted, however, that the reduction of target mRNA was already observed in the
Ribonucleoprotein complexes consisting of sRNAs, Hfq and RNAse E
SgrS is another Hfq-binding sRNA, the function of which has been demonstrated recently (see the review by Vanderpool [28] in this issue). SgrS is induced in response to accumulation of glucose-phosphate resulting in destabilization of the ptsG mRNA encoding the glucose transporter IICBGlc in an RNAse E-dependent fashion [19•, 29, 30]. The destabilization of ptsG mRNA is dependent on Hfq and the C-terminal scaffold region of RNAse E [24, 31•]. In addition, the rapid degradation of ptsG mRNA in
Translational repression is the primary event for gene silencing
As mentioned above, both SgrS and RyhB RNAs lead to the RNAse E-dependent degradation of target mRNAs and translational repression. An intriguing question is to what extent the RNAse E-dependent mRNA degradation contributes to the gene silencing by sRNAs. A recent study gave an answer to this question [37•]. It was shown that the synthesis of IICBGlc can be efficiently prevented by SgrS even in cells expressing the C-terminally truncated RNAse E in which the destabilization the ptsG mRNA no
Crucial base pairs for SgrS action
The requirement of base pairing for sRNA action has been shown experimentally, though not completely, in several cases such as OxyS–fhlA [38], DrsA–rpoS [39], RyhB–sodB [40], MicC–ompC [41] and MicA–ompA [42, 43]. Typically, studies have shown that mutations, in either a given sRNA or its target mRNA, that disrupt the predicted pairing decrease the regulation by the sRNA and that compensatory mutations restore the regulation. We carried out a more systematic mutational study to address the role
Role of Hfq
Hfq, originally identified as a host factor required for the in vitro replication of the RNA phage Qβ in E. coli [45], is an RNA binding protein extensively involved in the regulation of RNA metabolism [10]. The involvement of Hfq in sRNA functions was first recognized during the study on the mechanism of rpoS regulation by OxyS [46]. Subsequently, Hfq was shown to bind to and be necessary for the regulatory activity of a number of chromosomally encoded antisense sRNAs [10]. The protein
Role of membrane localization of target mRNAs
Another intriguing finding is that localization of target mRNA at the membrane is an important factor for SgrS action [31•, 48]. IICBGlc consists of an N-terminal domain, containing eight transmembrane segments, and a C-terminal cytoplasmic domain [49]. We found by using the ptsG–crp fusion genes that the region corresponding to the first two transmembrane stretches of ptsG is required for the rapid degradation of ptsG mRNA [31•]. A cytoplasmic ptsG–crp fusion mRNA became destabilized by SgrS
Conclusions
Studies on SgrS and RyhB sRNAs in the last few years have demonstrated that both RNAs share common features with other Hfq-binding sRNAs. Briefly, these two sRNAs are induced in response to a particular stress depending on a specific transcription factor and act through base-pairing with the ribosome-binding site of target mRNAs to downregulate gene expression. More importantly, studies on SgrS and RyhB have uncovered several novel mechanical features of sRNA action. Both RNAs destabilize
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
I thank Susan Gottesman for critical reading and comments on the review. I also thank Teppei Morita for preparing figures.
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