Evidence for an autonomous 5′ target recognition domain in an Hfq-associated small RNA

Abstract

The abundant class of bacterial Hfq-associated small regulatory RNAs (sRNAs) parallels animal microRNAs in their ability to control multiple genes at the posttranscriptional level by short and imperfect base pairing. In contrast to the universal length and seed pairing mechanism of microRNAs, the sRNAs are heterogeneous in size and structure, and how they regulate multiple targets is not well understood. This paper provides evidence that a 5′ located sRNA domain is a critical element for the control of a large posttranscriptional regulon. We show that the conserved 5′ end of RybB sRNA recognizes multiple mRNAs of Salmonella outer membrane proteins by ≥7-bp Watson-Crick pairing. When fused to an unrelated sRNA, the 5′ domain is sufficient to guide target mRNA degradation and maintain σ E -dependent envelope homeostasis. RybB sites in mRNAs are often conserved and flanked by 3′ adenosine. They are found in a wide sequence window ranging from the upstream untranslated region to the deep coding sequence, indicating that some targets might be repressed at the level of translation, whereas others are repressed primarily by mRNA destabilization. Autonomous 5′ domains seem more common in sRNAs than appreciated and might improve the design of synthetic RNA regulators.envelope stress | multiple targeting | noncoding RNA | porin S mall RNAs that act on trans-encoded target mRNAs by short base pairing are important posttranscriptional regulators in many organisms. The two most abundant classes to date are the microRNAs of eukaryotes and the Hfq-associated small regulatory RNAs (sRNAs) of gram-negative bacteria such as Escherichia coli and Salmonella (1). The ∼22-nt microRNAs use wellestablished mechanisms and machinery to repress mRNAs by short seed pairing within the 3′ UTR, and a single microRNA might regulate hundreds of genes in parallel (2).The bacterial sRNAs are also increasingly found to control multiple targets, although by binding the 5′ region of bacterial mRNAs (3). In fact, some sRNAs have an impact on dozens of genes under stress or altered growth conditions (4-7), with target sites being known for a subset of the regulated mRNAs. The Sm-like protein, Hfq, is required for intracellular stability and target annealing of the sRNAs (1, 8). Global analyses of Hfq-bound transcripts suggest an excess of potential targets over regulators (9, 10), further arguing that multiple targeting might be the general mode of sRNA action.Unlike the universal length and seed pairing of microRNAs, there are few common denominators for Hfq-dependent regulators. The sRNAs dramatically vary in size (50-250 nt) and secondary 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 . Most of the sRNAs analyzed to date inhibit translational initiation of targets by sequestering the Shine-Dalgarno (SD) or start codon (AUG) sequences of the ribosome binding site (RBS); how the recognition of these conserved RBS elements would ensure highly specific target...

Structural basis for RNA 3′-end recognition by Hfq

“…To characterize the RNA-binding properties of Salmonella typhimurium Hfq protein (St Hfq) we initially tested a series of RNA oligonucleotides containing an internal U-rich sequence found in RybB RNA, a well-characterized sRNA in Salmonella (22). RybB (Fig.…”

“…RybB-cP (nucleotides G1-U78; ref. 22) and R16-cP RNA were transcribed in vitro from a modified pSP64 plasmid containing a 3′-terminal hepatitis delta virus ribozyme that autocleaves cotranscriptionally, leaving a 2′-3′ cyclic phosphate on the target RNA. Target RNA was purified over denaturing polyacrylamide gels as described previously (32).…”

The ancestral SgrS RNA discriminates horizontally acquired Salmonella mRNAs through a single G-U wobble pair

“…3B); with respect to MFE, it is weaker than most Hfq-dependent sRNA-mRNA duplexes (44,49) including the well-studied SgrS-ptsG interaction (SI Appendix, Table S1). In fact, weaker duplexes have been found to allow posttranscriptional control [e.g., FnrS-folE (50) or ArcZ-sdaC (37)].…”

“…It was inspired by the ability of 22-nt microRNAs of eukaryotes to use their conserved seed region (nucleotides 2-7) to recognize mRNAs (46) and observations that Hfq-associated sRNAs carry conserved short regions that mediate target recognition (40,44,47,48). The latter includes pioneering work on SgrS, in which the work of Vanderpool and Gottesman (21) predicted targeting of ptsG mRNA by highly conserved nucleotides of SgrS, which are now referred to as the seed.…”

“…Successful seed pairing of small RNAs with targets has been proposed to largely depend on the thermodynamic stability of the RNA duplex that is formed (41,42). Accordingly, most target search algorithms, including the popular RNAhybrid , which was established with 10 targets of RybB sRNA (44).…”