Project description:Ribonuclease P (RNase P) is the only ribonuclease responsible for 5'-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, it is also essential for separation of pre-tRNAs from multiple polycistronic transcripts. Using RNA sequencing (RNA-seq), we show here that RNase P affects the abundance of ~44% of the expressed mRNAs demonstrating a much more widespread role in mRNA decay than previously thought. Furthermore, our data also demonstrate for the first time that the polyadenylation of transcripts by PAP I modulates RNase P-mediated mRNA decay as well as tRNA processing.
Project description:Abstract: RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes This SuperSeries is composed of the SubSeries listed below.
Project description:An experiment to identify the downstream targets of PatE, a prophage encoded AraC-like transcriptional regulator, in transcriptional activation of acid-resistance pathways of enterohemorrhagic Escherichia coli strain EDL933 using deletion and complementation strains (Delta3 and Delta3_1, respectively).
Project description:Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives compared to wild type (stabilome) in the stationary phase. The stabilome is an original dynamic transcriptome-based analysis to measure the rates of mRNA degradation at the genome scale. We have combined the analysis of stabilome with the steady state concentrations of mRNAs (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. The stabilome demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. This work allowed the roles of RNase R and PNPase in coordinating E. coli RNA metabolism to be discussed and PNPase to be identified as a central player of the degradation machinery in stationary phase.
Project description:Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives compared to wild type (stabilome) in the stationary phase. The stabilome is an original dynamic transcriptome-based analysis to measure the rates of mRNA degradation at the genome scale. We have combined the analysis of stabilome with the steady state concentrations of mRNAs (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. The stabilome demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. This work allowed the roles of RNase R and PNPase in coordinating E. coli RNA metabolism to be discussed and PNPase to be identified as a central player of the degradation machinery in stationary phase.
Project description:Housekeeping degradation of normal ribosomal RNA in eukaryotes is completed by the RNase T2 family of ribonucleases. This family of ribonucleases is found through eukaryotes, and the Arabidopsis RNase T2 which degrades ribosomal RNA is RNS2. A null mutant in RNS2, rns2-2, has higher levels of RNA, vacuolar accumulation of rRNA and constitutive autophagy. We used microarray to detail the global changes in gene expression in response to mutation of RNS2 (rns2-2) and identified key biological processes differentially regulated in response to this mutation.
Project description:The present study investigated the role(s) of RNase I (encoded by the rna gene) in Escherichia coli by comparative gene expression analysis of an rna mutant and the isogenic wild-type E. coli strain BW25113. The transcriptomic analysis aims to provide mechanistic insight into aberrant phenotypes observed in the RNase I-deficient mutant.
Project description:Propionate is an abundant carboxylic acid in nature. Microorganisms metabolize propionate aerobically via the 2-methylcitrate pathway. This pathway depends on a series of three reactions in the citric acid cycle that leads to the conversion of succinate to oxaloacetate. Interestingly, the gamma-proteobacterium Escherichia coli can use propionate as a carbon and electron source under oxic but not under anoxic conditions. The typical downregulation of the citric acid cycle under anoxic conditions is only partially responsible for the inability to use propionate under anoxic conditions since an arcA mutant shows very limited growth on propionate. RT-PCR and transcriptomic analysis revealed a post-transcriptional regulation of the prp-genecluster encoding the necessary enzymes for propionate metabolism. The polycistronic mRNA was hydrolyzed in the 3`-5` direction under anoxic conditions. This regulatory strategy is highly constructive because the last gene of the operon encodes the first enzyme of the propionate metabolism. Further analysis revealed that RNase R catalyzes the hydrolysis of the prp transcripts. Consequently, an rnr-deletion strain could metabolize propionate under anoxic conditions. To the best of our knowledge, this is the first study describing the influence of RNase R on the anaerobic metabolism of E. coli.