Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

Abstract

word count: 241 30Text word count: 6,690 31 32 33 34 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/221978 doi: bioRxiv preprint first posted online Nov. 19, 2017; ABSTRACT 35The mechanisms by which small RNA (sRNA) regulators select and prioritize 36 target mRNAs remain poorly understood, but serve to promote efficient responses to 37 environmental cues and stresses. We sought to uncover mechanisms that establish 38 regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced 39 under conditions of metabolic stress when sugar transport and metabolism are 40 CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/221978 doi: bioRxiv preprint first posted online Nov. 19, 2017; IMPORTANCE 58To survive, bacteria must respond rapidly to stress and simultaneously maintain 59 metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress 60 that arises from imbalanced sugar transport and metabolism. To coordinate the stress 61 response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic 62properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. 63To date, sRNA regulation of targets has largely been studied in the context of "one 64 sRNA-one target", and little is known about coordination of multi-gene regulons and 65 sRNA regulatory network structure. Here, we explore the molecular basis for regulatory 66 hierarchy in sRNA regulons. Our results reveal a complex interplay of factors, including 67 the number and location of sRNA binding sites on mRNA targets and the participation of 68 an RNA chaperone, that dictate prioritized regulation of targets to promote an efficient 69 response to stress. 70

Kinetic modeling reveals additional regulation at co-transcriptional level by post-transcriptional sRNA regulators

“…We next fit the models to two other SgrS targets, manX and purR. It has been established that ptsG is the primary target of SgrS, manX is a secondary target, and purR is a lower-priority target (Bobrovskyy et al, 2019). Consistently, we observed 78%, 53%, and 20% repression, respectively, for ptsG, manX, and purR at the protein level at 24 min under the same SgrS induction condition (Table S3).…”

“…However, the in vivo kinetic determinants that set the regulation hierarchy are largely unclear. A previous in vivo kinetic characterization of the sRNA target search and sRNA-mRNA co-degradation processes suggests that the in vivo binding affinity between specific sRNA-mRNA pairs can contribute to setting the regulatory hierarchy (Fei et al, 2015), whereas the in vitro binding affinity does not seem to correlate with the regulation hierarchy (Bobrovskyy et al, 2019). These observations suggest that in vivo target search and regulation kinetics may be collectively determined by complex molecular interactions and kinetic pathways that are difficult to fully recapitulate in vitro and therefore require in vivo characterization.…”

“…One characteristic feature of sRNA regulators is their ability to regulate multiple target mRNAs (Beisel and Storz, 2010;Bobrovskyy et al, 2019;Nitzan et al, 2017;Papenfort and Vogel, 2009). Previous studies have shown that the regulation of various targets by the same sRNA can exhibit a hierarchical pattern; i.e., certain targets are more effectively regulated than others (Jost et al, 2013;Levine et al, 2007).…”

RNA‐binding protein Hfq downregulates locus of enterocyte effacement‐encoded regulators independent of small regulatory RNA in enterohemorrhagic Escherichia coli

“…Hfq‐mediated translational regulation may conceivably be accomplished by three different mechanisms: (a) in canonical sRNA‐mediated regulation, sRNAs are responsible for regulating the expression and specificity of target mRNAs, and Hfq mediates the interaction between sRNAs and target mRNAs (Aiba, 2007; Vogel & Luisi, 2011); (b) sRNAs may only be responsible for target specificity, while Hfq functions in translational regulation and facilitation of sRNA–mRNA interactions (i.e., sRNA pairing on seed sequences other than the RBS of the target mRNA promotes the recruitment of Hfq to the ARN repeat near the RBS, and Hfq then inhibits translation initiation) (Azam & Vanderpool, 2018; Bobrovskyy et al., 2019; Desnoyers & Masse, 2012); (c) in sRNA‐independent regulation, Hfq participates in translational regulation and determination of the specificity of target mRNAs (i.e., Hfq directly and indirectly blocks ribosome access to target mRNAs by binding to ARN repeat sequences near the RBS of the mRNAs, and by forming an inhibitory hairpin near the RBS, respectively) (Chen & Gottesman, 2017; Ellis et al., 2015; Morita & Aiba, 2019; Salvail et al., 2013; Sonnleitner & Blasi, 2014). Our study showed that Hfq inhibits translation of grlA and ler via the third mechanism.…”

The Non-Coding Regulatory RNA Revolution in Archaea

“…Finally, in vivo quantitative measurements of sRNA-mediated regulation, such as those currently being made in the bacteria [46], are necessary to understand, at a system-level, how sRNA-based regulation is integrated within a cell's regulatory networks.…”