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Target Recognition Domain in an Hfq-Associated Small RNA

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Target Recognition Domain in an Hfq-Associated Small RNA Evidence for an autonomous 5′ target recognition domain in an Hfq-associated small RNA Kai Papenforta,b,1, Marie Bouvierb,1, Franziska Mikab,1, Cynthia M. Sharmaa,b, and Jörg Vogela,b,2 aInstitute for Molecular Infection Biology, University of Würzburg, D-97080 Würzburg, Germany; and bRNA Biology Group, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany Edited* by Carol A. Gross, University of California, San Francisco, CA, and approved October 18, 2010 (received for review July 6, 2010) The abundant class of bacterial Hfq-associated small regulatory maintained by selection (18) and often cluster in the 5′ sRNA re- RNAs (sRNAs) parallels animal microRNAs in their ability to control gion (6, 16, 20, 22–24). multiple genes at the posttranscriptional level by short and im- The ∼80-nt RybB sRNA was recently shown to accelerate perfect base pairing. In contrast to the universal length and seed mRNA decay of many major and minor outer membrane pro- pairing mechanism of microRNAs, the sRNAs are heterogeneous in teins (OMPs) in Salmonella and E. coli (25, 26). RybB is acti- E size and structure, and how they regulate multiple targets is not vated by the alternative σ factor when excessive OMP synthesis well understood. This paper provides evidence that a 5′ located or envelope damage causes periplasmic folding stress (25–28). σE sRNA domain is a critical element for the control of a large post- As such, RybB is a major facilitator of -directed global OMP transcriptional regulon. We show that the conserved 5′ end of RybB repression (25, 29) and is required for envelope homeostasis and σE sRNA recognizes multiple mRNAs of Salmonella outer membrane feedback regulation of (25, 27). ≥ – Pulse expression of Salmonella RybB reduces the half-lives of proteins by 7-bp Watson Crick pairing. When fused to an unre- ≥ ∼ lated sRNA, the 5′ domain is sufficient to guide target mRNA deg- stable omp mRNAs from of 10 min to 1 min (25), and both radation and maintain σE-dependent envelope homeostasis. RybB RybB and its putative targets interact with Hfq (10), all of which sites in mRNAs are often conserved and flanked by 3′ adenosine. predicts direct regulation by RNA interactions. Biocomputational algorithms failed to predict statistically significant RybB pairing They are found in a wide sequence window ranging from the up- with the RBS of targets (25), however, and the only known RybB stream untranslated region to the deep coding sequence, indicating site thus far was discovered in the coding sequence of Salmonella that some targets might be repressed at the level of translation, ompN mRNA (17). whereas others are repressed primarily by mRNA destabilization. ′ ′ Here, we report genetic evidence that the conserved 5 end of Autonomous 5 domains seem more common in sRNAs than appre- RybB constitutes an autonomous multiple-target binding domain ciated and might improve the design of synthetic RNA regulators. used to select many omp mRNAs by short (≥7 bp) Watson–Crick pairing. This domain is essential and sufficient for target repression envelope stress | multiple targeting | noncoding RNA | porin and adjusting σE activity. RybB sites in omp mRNAs are associated with a unique 3′ adenosine signal and are located in a broad window mall RNAs that act on trans-encoded target mRNAs by short ranging from 5′ located stability elements into the deep coding Sbase pairing are important posttranscriptional regulators in sequence, suggesting varying contributions of translational in- many organisms. The two most abundant classes to date are the hibition and mRNA decay to target repression. An autonomous microRNAs of eukaryotes and the Hfq-associated small regula- 5′ domain might be more common in sRNAs than appreciated and tory RNAs (sRNAs) of gram-negative bacteria such as Escher- also governs the prototypical MicF-ompF regulation. ichia coli and Salmonella (1). The ∼22-nt microRNAs use well- established mechanisms and machinery to repress mRNAs by Results short seed pairing within the 3′ UTR, and a single microRNA OMP Depletion by Constitutive RybB Expression. RybB effects have might regulate hundreds of genes in parallel (2). been studied at the omp mRNA level following short expression of The bacterial sRNAs are also increasingly found to control the sRNA (25, 26). To assay regulation at the protein level, we multiple targets, although by binding the 5′ region of bacterial used the constitutive expression plasmid pPL-RybB (17), which mRNAs (3). In fact, some sRNAs have an impact on dozens of genes confers equivalent RybB levels throughout growth (Fig. 1) and under stress or altered growth conditions (4–7), with target sites approximately fivefold more than the chromosomal rybB gene being known for a subset of the regulated mRNAs. The Sm-like after σE induction (SI Appendix, Fig. S1). We observed full de- protein, Hfq, is required for intracellular stability and target an- pletion of three 30- to 35-kDa proteins—OmpA, OmpC, and nealing of the sRNAs (1, 8). Global analyses of Hfq-bound tran- OmpD—known as the most abundant Salmonella porins during scripts suggest an excess of potential targets over regulators (9, 10), regular growth (Fig. 1). Depletion of the fourth major porin, further arguing that multiple targeting might be the general mode of OmpF, was confirmed using strains with individual omp gene sRNA action. disruptions (SI Appendix, Fig. S2). In contrast, there was no effect Unlike the universal length and seed pairing of microRNAs, on the OmpX porin (Fig. 1) whose mRNA is refractory to RybB in there are few common denominators for Hfq-dependent regu- Salmonella (25), suggesting that repression was specific and not lators. The sRNAs dramatically vary in size (50–250 nt) and sec- caused by a general block of OMP biogenesis. Because constitu- ondary structure (11), and sRNA-mRNA interactions range from >30-bp duplexes in MicF-ompF or Spot42-galK (12, 13) to only 6 bps that are critical in SgrS-ptsG (14). Most of the sRNAs analyzed Author contributions: K.P., M.B., F.M., C.M.S., and J.V. designed research; K.P., M.B., F.M., to date inhibit translational initiation of targets by sequestering the and C.M.S. performed research; K.P., M.B., F.M., C.M.S., and J.V. analyzed data; and K.P. Shine–Dalgarno (SD) or start codon (AUG) sequences of the ri- and J.V. wrote the paper. bosome binding site (RBS); how the recognition of these conserved The authors declare no conflict of interest. RBS elements would ensure highly specific target selection is little *This Direct Submission article had a prearranged editor. understood (15). Interestingly, there are a few recent examples of Freely available online through the PNAS open access option. sRNA binding upstream or downstream of SD/AUG sequences 1K.P., M.B., and F.M. contributed equally to this work. – 2 (16 21), suggesting that the mRNA window for target repression To whom correspondence should be addressed. E-mail: [email protected]. GENETICS could be broader than the RBS. Emerging evidence suggests that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. those sRNA nucleotides interacting with multiple targets might be 1073/pnas.1009784107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1009784107 PNAS | November 23, 2010 | vol. 107 | no. 47 | 20435–20440 Downloaded by guest on September 23, 2021 pJV300 pPL -RybB the fusion proteins was determined by Western blot analysis (Fig. 0.5 1.5 2.0 3.4 0.5 1.5 2.0 3.6 OD600 2C, Bottom). RybB Wild-type RybB repressed all target fusions (Fig. 2C, lane 2), predicting that the cloned mRNA regions contained a RybB tar- 5S get site; these repressions were specific because RybB did not 175 regulate gfp alone or an ompX::gfp control fusion. As with the 83 major OMPs, the RybBΔ1–9 variant did not regulate any of the fusions (Fig. 2C, lane 3). In contrast, R16TMA and R16TOM 62 down-regulated all fusions (Fig. 2C, lanes 4 and 5) to the same 47 degree as WT RybB; we attributed these regulations to R16 be- cause the parental control RNAs (TMA and TOM) alone had C no impact on reporter activity (Fig. 2C, lanes 6 and 7). Together, major 32 D these results narrowed down the sequence space for the predic- A porins tion of RybB binding. RybB Target Sites in omp mRNAs. We used the RNAhybrid algo- 25 rithm (31) to predict RNA duplexes of R16 with the above cloned 5′ target mRNA fragments. Fig. 3A shows the highest scoring interaction for each target, all of which have a change in free −1 16 energy (ΔG°) in a −24.2 to −18.8 kcal/mol range, similar to the C - porin known RybB-ompN pairing (included for comparison) yet far F − − −1 D more favorable than the 13 to 8 kcal/mol range expected for A random RNA pairing (15). Intriguingly, each interaction involves - OmpX 5′ terminal pairing of RybB, commonly of seven or more con- 1 2 3 4 5 6 7 8 secutive Watson–Crick pairs. Fig. 1. Constitutive RybB expression causes porin depletion in Salmonella. The RybB target sites are strikingly diverse in relative mRNA RNA and protein samples were prepared from WT Salmonella carrying plas- position (Fig. 3A). Canonical binding sites overlapping SD/AUG − mids pJV300 (empty vector; lanes 1–4) or pPL-RybB (lanes 5–8) after growth to must fall within a 16 to +3 window on mRNA (+1 is A of the start exponential phase (OD600 of 0.5) or stationary phase (OD600 of 1.5, 2, or 3.4). codon AUG); only tsx would be targeted so.
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