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Global analysis of mRNA decay intermediates in Bacillus subtilis wild-type and polynucleotide phosphorylase-deletion strains

ABSTRACT: Messenger RNA decay in Bacillus subtilis is accomplished by a combination of exoribonucleases and endoribonucleases. Intermediates in the decay process have not been readily detectable, and previous studies on mRNA decay have used a handful of highly expressed transcripts as models. Here, we use RNA‐Seq analysis to probe mRNA turnover globally. A significant fraction of messages showed differential accumulation of RNA fragments that mapped near the 5′ or 3′ end of the coding sequence, consistent with initiation of decay from either the 5′ end or from an internal cleavage site. Patterns of mRNA decay in the wild type were compared with patterns in a mutant strain lacking polynucleotide phosphorylase (PNPase), which is considered the major 3′ exonuclease activity in mRNA decay and which is one of four known 3′ exonucleases in B. subtilis. The results showed a striking dependence on PNPase for mRNA turnover in many cases, suggesting specificity in the ability of 3′ exonucleases to degrade from 3′‐hydroxyl termini. RNA‐Seq data demonstrated a sharp decrease in expression of Sigma D in the PNPase‐deletion strain. Reduction in sigD regulon expression explained the chain growth phenotype of the PNPase mutant and also predicted a defect in swarming motility.

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE120050 | GEO | 2018/09/18

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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Global analysis of mRNA decay intermediates in Bacillus subtilis wild-type and polynucleotide phosphorylase-deletion strains

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