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Co-immunoprecipitation of the small DUF1127 protein RSP_6037xFLAG and pRK0557_3xFLAG for RNA interaction

ABSTRACT: Many protein domains are conserved among different classes and are found in numerous species, but their function remains obscure. Data bases list more than 17,000 DUF1127 proteins but to date no biological function could be assigned to any of these proteins. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in the maturation of the CcsR sRNAs that are processed from the 3´ UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 also affects the abundance of several other sRNAs but also of tRNAs, rRNAs and several mRNAs. Co-immunoprecipitation revealed direct binding of CcaF1 to many cellular RNAs of different type and an effect of CcaF1 on the stability of selected RNAs was demonstrated. Thus, the widely distributed DUF1127 domain can mediate RNA-binding and affect stability of its binding partners and consequently the bacterial transcriptome.

ORGANISM(S): Cereibacter sphaeroides 2.4.1

PROVIDER: GSE145045 | GEO | 2021/02/19

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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Co-immunoprecipitation of the small DUF1127 protein RSP_6037xFLAG and pRK0557_3xFLAG for RNA interaction

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