Dataset Information

Impact of PNPase on the transcriptome of Rhodobacter sphaeroides and its cooperation with RNase III and RNase E.

ABSTRACT:

Background

The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3'-to-5' exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5'-to-3' end direction which has a destabilizing effect on the prolonged RNA molecule. RNA degradation is often initiated by an endonucleolytic cleavage, followed by exoribonucleolytic decay from the new 3' end.

Results

The PNPase mutant from the facultative phototrophic Rhodobacter sphaeroides exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. The transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. Moreover, we globally identified and compared differential RNA 3' ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants for the first time in a Gram-negative organism. The genome wide RNA 3' end analysis revealed that 885 3' ends are degraded by PNPase. A fair percentage of these RNA 3' ends was also identified at the same genomic position in RNase E or RNase III mutant strains.

Conclusion

The PNPase has a major influence on RNA processing and maturation and thus modulates the transcriptome of R. sphaeroides. This includes sRNAs, emphasizing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The global 3' end analysis indicates a sequential RNA processing: 5.9% of all RNase E-dependent and 9.7% of all RNase III-dependent RNA 3' ends are subsequently degraded by PNPase. 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 license.

SUBMITTER: Spanka DT

PROVIDER: S-EPMC7866481 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Impact of PNPase on the transcriptome of Rhodobacter sphaeroides and its cooperation with RNase III and RNase E.

Spanka Daniel-Timon DT Reuscher Carina Maria CM Klug Gabriele G

BMC genomics 20210206 1

<h4>Background</h4>The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3'-to-5' exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5'-to-3' end direction which has a destabilizing effect on the pr ...[more]

PMID: 33549057

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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.

Project description:Rhodobacter sphaeroides 2.4.1 is a facultative photosynthetic anaerobe that grows by anoxygenic photosynthesis under anaerobic-light conditions. Changes in energy generation pathways under photosynthetic and aerobic respiratory conditions are primarily controlled by oxygen tensions. In this study, we performed time series microarray analyses to investigate transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration. Major changes in gene expression profiles occurred in the initial 15 min after the shift from anaerobic-light to aerobic-dark conditions, with changes continuing to occur up to 4 hours postshift. Those genes whose expression levels changed significantly during the time series were grouped into three major classes by clustering analysis. Class I contained genes, such as that for the aa3 cytochrome oxidase, whose expression levels increased after the shift. Class II contained genes, such as those for the photosynthetic apparatus and Calvin cycle enzymes, whose expression levels decreased after the shift. Class III contained genes whose expression levels temporarily increased during the time series. Many genes for metabolism and transport of carbohydrates or lipids were significantly induced early during the transition, suggesting that those endogenous compounds were initially utilized as carbon sources. Oxidation of those compounds might also be required for maintenance of redox homeostasis after exposure to oxygen. Genes for the repair of protein and sulfur groups and uptake of ferric iron were temporarily upregulated soon after the shift, suggesting they were involved in a response to oxidative stress. The flagellar-biosynthesis genes were expressed in a hierarchical manner at 15 to 60 min after the shift. Numerous transporters were induced at various time points, suggesting that the cellular composition went through significant changes during the transition from anaerobic photosynthesis to aerobic respiration. Analyses of these data make it clear that numerous regulatory activities come into play during the transition from one homeostatic state to another.

Dataset Information

Impact of PNPase on the transcriptome of Rhodobacter sphaeroides and its cooperation with RNase III and RNase E.

Background

Results

Conclusion

Publications

Impact of PNPase on the transcriptome of Rhodobacter sphaeroides and its cooperation with RNase III and RNase E.

Similar Datasets

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