A minimal base‐pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA

Escherichia coli SgrS is an Hfq‐binding small RNA that is induced under glucose‐phosphate stress to cause translational repression and RNase E‐dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31‐nt‐long stretch in the 3′ region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre‐annealed with ptsG mRNA by a high‐temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base‐pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base‐pairing region is sufficient to inhibit ptsG translation in the context of full‐length SgrS in vivo. We conclude that SgrS 168–181 is a minimal base‐pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high‐temperature pre‐treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin

The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)‐regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed‐forward loop mechanism related to iron physiology and colicin sensitivity.     

microRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA‐binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA‐independent manner, thereby repressing translation.     

PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes

In Trypanosoma brucei, most mitochondrial mRNAs undergo internal changes by RNA editing and 3′ end modifications. The temporally separated and functionally distinct modifications are manifested by adenylation prior to editing, and by post‐editing extension of a short A‐tail into a long A/U‐heteropolymer. The A‐tail stabilizes partially and fully edited mRNAs, while the A/U‐tail enables mRNA binding to the ribosome. Here, we identify an essential pentatricopeptide repeat‐containing RNA binding protein, kinetoplast polyadenylation factor 3 (KPAF3), and demonstrate its role in protecting pre‐mRNA against degradation by the processome. We show that KPAF3 recruits KPAP1 poly(A) polymerase to the 3′ terminus, thus leading to pre‐mRNA stabilization, or decay depending on the occurrence and extent of editing. In vitro, KPAF3 stimulates KPAP1 activity and inhibits mRNA uridylation by RET1 TUTase. Our findings indicate that KPAF3 selectively directs pre‐mRNA toward adenylation rather than uridylation, which is a default post‐trimming modification characteristic of ribosomal and guide RNAs. As a quality control mechanism, KPAF3 binding ensures that mRNAs entering the editing pathway are adenylated and, therefore, competent for post‐editing A/U‐tailing and translational activation.     

Antisense regulation by transposon‐derived RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus

We report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In Sulfolobus solfataricus, the transposon‐derived paralogous RNAs, RNA‐257_1–4, show extended complementarity to the 3′ UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation results in decreased RNA‐257₁ and increased 1183 mRNA levels. Correspondingly, the 1183 mRNA is faster degraded in vitro upon duplex formation with RNA‐257₁. Insertion of the 1183 3′ UTR downstream of the lacS gene results in strongly reduced lacS mRNA levels in transformed cells, indicating that antisense regulation can function in trans.