Abstract
Small regulatory RNAs (sRNAs) are an important class of bacterial post-transcriptional regulators that control numerous physiological processes, including stress responses. In Gram-negative bacteria including Escherichia coli, the RNA chaperone Hfq binds many sRNAs and facilitates pairing to target transcripts, resulting in changes in mRNA transcription, translation, or stability. Here, we report that poly(A) polymerase (PAP I), which promotes RNA degradation by exoribonucleases through the addition of poly(A) tails, has a crucial role in the regulation of gene expression by Hfq-dependent sRNAs. Specifically, we show that deletion of pcnB, encoding PAP I, paradoxically resulted in an increased turnover of certain Hfq-dependent sRNAs, including RyhB. RyhB instability in the pcnB deletion strain was suppressed by mutations in hfq or ryhB that disrupt pairing of RyhB with target RNAs, by mutations in the 3′ external transcribed spacer of the glyW-cysT-leuZ transcript (3′ETSLeuZ) involved in pairing with RyhB, or an internal deletion in rne, which encodes the endoribonuclease RNase E. Finally, the reduced stability of RyhB in the pcnB deletion strain resulted in impaired regulation of some of its target mRNAs, specifically sodB and sdhCDAB. Altogether our data support a model where PAP I plays a critical role in ensuring the efficient decay of the 3′ETSLeuZ. In the absence of PAP I, the 3′ETSLeuZ transcripts accumulate, bind Hfq, and pair with RyhB, resulting in its depletion via RNase E-mediated decay. This ultimately leads to a defect in RyhB function in a PAP I deficient strain.
Highlights
IntroductionSmall regulatory RNAs (sRNAs) are transcripts ranging in size from 50 to 300 nucleotides (nt) that have been shown to regulate nearly every aspect of bacterial behavior and physiology including virulence (Toledo-Arana et al 2009; Gripenland et al 2010; Felden et al 2011; Koo et al 2011; Bardill and Hammer 2012; Hébrard et al 2012; Mann et al 2012; Rutherford and Bassler 2012; Caldelari et al 2013; Holmqvist and Wagner 2017), biofilm development (Thomason et al 2012; Jørgensen et al 2013; Zhao et al 2013; Parker et al 2017), antibiotic resistance (Parker and Gottesman 2016; Zhang et al 2017; Felden and Cattoir 2018), and metabolism (Durand and Storz 2010; Gimpel et al 2010; Richards and Vanderpool 2011; Salvail and Massé 2012; Bobrovskyy and Vanderpool 2013; McClure et al 2013; Mandin et al 2016; Pannuri et al 2016; Gonzalez et al 2017)
SRNAs can positively regulate gene expression by base-pairing with a mRNA as it is being transcribed, preventing intramolecular base-pairing in the 5′ untranslated region (5′-UTR) that would otherwise lead to transcription attenuation (Sedlyarova et al 2016)
Hfq promotes base-pairing with the sdhCDAB mRNA, blocking expression of succinate dehydrogenase complex and leading to an inability of a fur mutant to grow on succinate as the sole carbon source (Massé and Gottesman 2002)
Summary
Small regulatory RNAs (sRNAs) are transcripts ranging in size from 50 to 300 nucleotides (nt) that have been shown to regulate nearly every aspect of bacterial behavior and physiology including virulence (Toledo-Arana et al 2009; Gripenland et al 2010; Felden et al 2011; Koo et al 2011; Bardill and Hammer 2012; Hébrard et al 2012; Mann et al 2012; Rutherford and Bassler 2012; Caldelari et al 2013; Holmqvist and Wagner 2017), biofilm development (Thomason et al 2012; Jørgensen et al 2013; Zhao et al 2013; Parker et al 2017), antibiotic resistance (Parker and Gottesman 2016; Zhang et al 2017; Felden and Cattoir 2018), and metabolism (Durand and Storz 2010; Gimpel et al 2010; Richards and Vanderpool 2011; Salvail and Massé 2012; Bobrovskyy and Vanderpool 2013; McClure et al 2013; Mandin et al 2016; Pannuri et al 2016; Gonzalez et al 2017). Many sRNAs regulate these processes by recognizing and binding specific target mRNAs through base-pairing resulting in changes in mRNA transcription, translation, or stability depending on the nature of this interaction. Negative regulation of gene expression can be achieved by sRNA-induced RNase E mediated decay of mRNAs (Pfeiffer et al 2009; Bandyra et al 2012). SRNAs can positively regulate gene expression by base-pairing with a mRNA as it is being transcribed, preventing intramolecular base-pairing in the 5′ untranslated region (5′-UTR) that would otherwise lead to transcription attenuation (Sedlyarova et al 2016). In Escherichia coli and other Gram-negative bacteria, many sRNAs encoded in trans require the RNA-binding protein chaperone Hfq to function
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