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Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments

ABSTRACT: In many gram-negative and some gram-positive bacteria small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism, and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA-mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase uniquely contributes to the degradation of specific mRNA cleavage products, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of a sRNA eliminated the requirement of PNPase for sRNA stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing mediated decay.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE125368 | GEO | 2019/07/08

REPOSITORIES: GEO

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Project description:The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the degradosome, the PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay since it acts as a 3’- to 5’-exoribonuclease. Furthermore PNPase can catalyze the reverse reaction by elongating RNA molecules in 5’- to 3’-end direction which finally has a destabilizing effect on the prolonged RNA molecule. RNA degradation can be initiated by an endonucleolytic cleavage and then by further promoted by exoribonucleolytic decay. In this study, we analyzed the importance of the Rhodobacter sphaeroides PNPase regarding the physiology and sRNA homeostasis. Moreover, we developed a pipeline to globally identify differential RNA 3’-ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants. The Rhodobacter sphaeroides PNPase mutant exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. Furthermore, the transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, the PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. The RNA 3’-end analysis reveals that 82% of all differential 3’-ends are enriched in the pnp mutant and are mostly located in coding sequences or untranslated regions (UTR). A fair percentage of these ends was also identified as differential RNA 3’-end in RNase E and RNase III mutant strains. The PNPase has a major influence in RNA processing and maturation and thus modulates the transcriptome. This includes regulatory sRNAs, stressing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The 3’-end analysis indicates a sequential processing of UTR- and non-UTR derived sRNAs by several ribonucleases. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5’/3’-ends. It is publicly available on GitHub and is distributed under ICS licence.

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Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments

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