Multiple factors dictate target selection by Hfq-binding small RNAs

Abstract

Hfq-binding small RNAs (sRNAs) in bacteria modulate the stability and translational efficiency of target mRNAs through limited base-pairing interactions. While these sRNAs are known to regulate numerous mRNAs as part of stress responses, what distinguishes targets and non-targets among the mRNAs predicted to base pair with Hfq-binding sRNAs is poorly understood. Using the Hfq-binding sRNA Spot 42 of Escherichia coli as a model, we found that predictions using only the three unstructured regions of Spot 42 substantially improved the identification of previously known and novel Spot 42 targets. Furthermore, increasing the extent of base-pairing in single or multiple base-pairing regions improved the strength of regulation, but only for the unstructured regions of Spot 42. We also found that non-targets predicted to base pair with Spot 42 lacked an Hfq-binding site, folded into a secondary structure that occluded the Spot 42 targeting site, or had overlapping Hfq-binding and targeting sites. By modifying these features, we could impart Spot 42 regulation on non-target mRNAs. Our results thus provide valuable insights into the requirements for target selection by sRNAs.

The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism

“…In agreement with this observation base-pairing with targets are rarer and only observed for 2 targets (galK and pirin). This is in agreement with results from Beisel et al 15 Using 3 unstructured regions (the 5' single stranded region, the 5' hairpin and the single-stranded region separating the hairpins) as input during computational target identification, they improved identification of direct targets, compared to when using the full-length sequence of Spot 42. In summary, highly conserved nucleotide Figure 2.…”

“…Fig. 3 is based on data from Møller et al, 1 Hansen et al, 4 Beisel and Storz, 14 and Beisel et al 15 Aeromonadales, Alteromonadales, Enterobacteriales and Vibrionales share the same last common ancestor, whereas Chromatiales does not, which suggest that spf was introduced into Chromatiales by lateral transfer by a donor from the clade marked by an arrow. We made a consensus secondary structure model of Spot 42 based on all known spf sequences and compared this to a schematically figure showing potential basepairing between Spot 42 and known mRNA targets.…”

“…This is in agreement with an earlier study by Beisel et al which suggested that the strength of Spot 42 regulation is directly dependent on the number of nucleotides and the number of highly conserved structural regions which are involved in base-pairing between Spot 42 and its target. 15 It is intriguing to us that although Spot 42 was discovered more than 40 years ago, there are still many unanswered questions. As more sequence data are being produced from high-throughput sequencing techniques and better tools and search algorithms are being developed, the known natural distribution of spf will certainly expand to new orders, families and genera (and perhaps phyla).…”

“…Interactions between Spot 42 and galK mRNA has been determined using structure probing, 1 whereas potential base-pairing to other targets is based on bioinformatics predictions followed by experimental work. 4,14,15 Figure 3 shows schematically potential base-pairing between Spot 42 and experimentally verified mRNA targets for the following genes: galK, pirin, fucI, xylF, sthA, gltA, srlA, nanC, paaK, ascF, caiA, fucP, atoD, puuE and nanT. Interestingly, for all except 2 genes (i.e., sthA and fucP) the most conserved region of the 5' hairpin (i.e., pos.…”

“…Compared to when using fulllength Spot 42 sequence as input the target identification was improved and additional targets were revealed. 15 The target analysis combined with assays of reporter fusions identified 7 novel Spot 42 mRNA targets, all involved in catabolite repression. Mutational analysis showed that the interactions of the 3 conserved regions of Spot 42 are critical in target regulation and that regulation through multiple conserved regions of Spot 42 as well as increased base-pairing in these regions strengthen the target regulation.…”