Small RNA Modules Confer Different Stabilities and Interact Differently with Multiple Targets

<div><p>Bacterial Hfq-associated small regulatory RNAs (sRNAs) parallel animal microRNAs in their ability to control multiple target mRNAs. The small non-coding MicA RNA represses the expression of several genes, including major outer membrane proteins such as <em>omp</em>A, <em>tsx</em> and <em>ecn</em>B. In this study, we have characterised the RNA determinants involved in the stability of MicA and analysed how they influence the expression of its targets. Site-directed mutagenesis was used to construct MicA mutated forms. The 5′linear domain, the structured region with two stem-loops, the A/U-rich sequence or the 3′ poly(U) tail were altered without affecting the overall secondary structure of MicA. The stability and the target regulation abilities of the wild-type and the different mutated forms of MicA were then compared. The 5′ domain impacted MicA stability through an RNase III-mediated pathway. The two stem-loops showed different roles and disruption of stem-loop 2 was the one that mostly affected MicA stability and abundance. Moreover, STEM2 was found to be more important for the <em>in vivo</em> repression of both <em>omp</em>A and <em>ecn</em>B mRNAs while STEM1 was critical for regulation of <em>tsx</em> mRNA levels. The A/U-rich linear sequence is not the only Hfq-binding site present in MicA and the 3′ poly(U) sequence was critical for sRNA stability. PNPase was shown to be an important exoribonuclease involved in sRNA degradation. In addition to the 5′ domain of MicA, the stem-loops and the 3′ poly(U) tail are also shown to affect target-binding. Disruption of the 3′U-rich sequence greatly affects all targets analysed. In conclusion, our results have shown that it is important to understand the “sRNA anatomy” in order to modulate its stability. Furthermore, we have demonstrated that MicA RNA can use different modules to regulate its targets. This knowledge can allow for the engineering of non-coding RNAs that interact differently with multiple targets.</p> </div

Mutagenesis of the A/U-rich domain, a Hfq-binding site in MicA.

<p>(A) Northern blot analysis of MicA in Δ<i>mic</i>A cells expressing <i>in trans</i> either the wild-type MicA (from the pMicA-WT plasmid) or a MicA variant with the A/U-rich domain mutated to a C-rich sequence (from the pMicA-hfq plasmid). Plasmid pMicA-WT was also used to transform a deletion strain of <i>hfq</i>. A smaller form of MicA (designated MicA∗) is only clearly observed in the absence of Hfq; this fragment had been previously identified <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Andrade2" target="_blank">[13]</a>. A size marker is shown on the left of the gel. The riboprobe used to detect MicA, cross-reacts with a nonspecific band, (that is also detected on the Δ<i>mic</i>A strain, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866.s006" target="_blank">Figure S6</a>) that was here used as loading control. A more stringent washing step eliminates this band without affecting the MicA signal, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Andrade1" target="_blank">[12]</a>. RNA was extracted from stationary phase cultures. (B) Mutagenesis of the A/U-rich domain of MicA to a C-rich sequence affects the Hfq binding ability to this small RNA. The gel mobility shift assay was performed with a constant amount of radiolabelled MicA-WT or MicA-hfq variant as RNA substrates and increasing amounts of purified Hfq protein, as indicated in the figure. The free RNA and the Hfq-RNA complexes are indicated. The gels were then dried and exposed to a PhosphorImager screen and quantified using ImageQuant software. The results were plot using SigmaPlot software and binding curves were fit. Filled circles represent MicA-WT and open circles represent MicA-hfq variant.</p

Construction of MicA mutants.

<p>Nucleotide sequence and secondary structure of the <i>E. coli</i> wild-type MicA is shown on top. The proposed modular domain organization of MicA is indicated. MicA sequence and structure is conserved in several enterobacteria as analysed by the CLUSTALW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Chenna1" target="_blank">[65]</a> and the RNAalifold software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Bernhart1" target="_blank">[66]</a>. The model structures predicted with mfold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Zuker1" target="_blank">[18]</a> for the different MicA mutants are schematized. Mutagenesis of the different domains was designed to not disturb the overall secondary structure of the MicA RNA. Nucleotides changes are indicated by arrows.</p

Plasmids used in this work.

<p>Plasmids used in this work.</p

Mutagenesis of the 5′ linear domain of MicA.

<p>Northern blot analysis of MicA in Δ<i>mic</i>A cells or its isogenic derivative lacking RNase III (Δ<i>mic</i>A Δ<i>rnc</i>), expressing <i>in trans</i> either the wild-type MicA (from the pMicA-WT plasmid) or the 5′ mutated MicA variant (from the pMicA-5′mut plasmid). RNA was extracted from stationary phase cultures and MicA stability was measured as described in <i>Material and Methods</i>.</p

Strains used in this work.

<p>Strains used in this work.</p

Mutagenesis of MicA stem-loops.

<p>(A) Northern blot analysis of MicA in Δ<i>mic</i>A cells expressing <i>in trans</i> either the wild-type MicA (from the pMicA-WT plasmid) or the stem-loops mutated MicA variants (from the pMicA-STEM1 or pMicA-STEM2 plasmids). When expressing the MicA-WT it is possible to visualise two lower molecular weight bands (<60 nts) that were previously identified in work performed in <i>Salmonella</i> to correspond to breakdown products of the duplex MicA-target mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Viegas1" target="_blank">[41]</a>. (B) Impact of the disruption of both stem-loops (MicA-STEM1_2 variant) on MicA stability. Plasmid pMicA-STEM1_2 was used to transform Δ<i>mic</i>A cells and its isogenic derivatives lacking PNPase (Δ<i>mic</i>A <i>pnp</i>) or RNase III (Δ<i>mic</i>A Δ<i>rnc</i>). (C) Northern blot analysis of the chromosomally encoded MicA or the MicA-WT expressed from plasmid, comparing the stability pattern in cells expressing or not PNPase. RNA was extracted from stationary phase cultures.</p

Mutagenesis of the 3′ end U-rich domain of MicA.

<p>(A) Effect of mutations in the 3′end U-rich linear sequence in the stability of the MicA RNA. Northern blot analysis of MicA in Δ<i>mic</i>A cells expressing <i>in trans</i> the wild-type MicA (from the pMicA-WT) or the mutated 3′ end variants (from the pMicA-3′mut1 or pMicA-3′mut2 plasmids). Read-through bands are indicated by the symbol (¶). Two different sized forms of MicA can be detected and are marked with arrows on the side of the gel. (B) Northern blot analysis of MicA in Δ<i>mic</i>A cells or its derivative isogenic mutants lacking either PNPase (Δ<i>mic</i>A <i>pnp</i>) or Poly(A) polymerase I (Δ<i>mic</i>A Δ<i>pcn</i>B) expressing <i>in trans</i> either the mutated MicA-3′mut1 or the MicA-3′mut2 variant. RNA was extracted from stationary phase cultures. Upon hybridization of the membrane, a nonspecific band is observed and was here used as loading control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052866#pone.0052866-Andrade1" target="_blank">[12]</a>.</p

Primers used in this work.

<p>Nucleotide changes are indicated in small capitals;</p><p>T7 promotor sequence is underlined.</p><p>“+” precedes LNA-modified nucleotides.</p

RliB is a substrate for PNPase.

<p>A) <i>In vitro</i> PNPase activity assay. A 37 nt short P³²-labeled substrate RNA (RNA37*) was incubated alone (lane 1) or with 200 nM purified PNPase (lane 2). Three types of non-labeled competitor RNAs were then added: non-labeled RliB (lanes 3, 100 nM; lane 4, 200 nM; lane 5, 400 nM), non-labeled control RNA RsaA (lane 6, 100 nM; lane 7, 200 nM; lane 8, 400 nM) and non-labeled RNA37 substrate (lane 9, 100 nM; lane 10, 200 nM; lane 11, 400 nM). B) <i>In vitro</i> PNPase-mediated processing of RliB. On the left are indicated ladders T1 (RNase T1 hydrolysis) and L (alkaline hydrolysis). The full length P³²-5′ labeled RliB (RliB*) was incubated with increasing concentrations of purified PNPase (lane 1, no PNPase; lane 2, 25 nM; lane 3, 100 nM; lane 4, 400 nM). Competition experiments were performed in the presence of 400 nM non-labelled RliB (lane 5). C) <i>In vivo</i> PNPase-mediated processing of RliB. Northern blot performed on the total RNAs extracted from the <i>L. monocytogenes</i> EGD-e wild type bacteria (WT), strain deleted for <i>pnpA</i> (Δ<i>pnpA</i>), complemented strain (Δ<i>pnpA-pnpA</i>) and strain deleted for RliB (Δ<i>rliB</i>). Expression of RliB was detected by the P³² labeled <i>in vitro</i> transcribed RNA probe complementary to the full length RliB. The membrane was reprobed with P³² labeled <i>in vitro</i> transcribed RNA probe complementary to the tmRNA.</p