Dataset Information

Escherichia coli cold-shock gene profiles in response to over-expression/deletion of CsdA, RNase R and PNPase and relevance to low-temperature RNA metabolism.

ABSTRACT: Cold-shock response is elicited by the transfer of exponentially growing cells from their optimum temperature to a significantly lower growth temperature and is characterized by the induction of several cold-shock proteins. These proteins, which presumably possess a variety of different activities, are critical for survival and continued growth at low temperature. One of the main consequences of cold shock is stabilization of the secondary structures in nucleic acids leading to hindrance of RNA degradation. Cold-shock proteins, such as RNA helicase CsdA, and 3'-5' processing exoribonucleases, such as PNPase and RNase R, are presumably involved in facilitating the RNA metabolism at low temperature. As a step toward elucidating the individual contributions of these proteins to low-temperature RNA metabolism, the global transcript profiles of cells lacking CsdA, RNase R and PNPase proteins as well as cells individually over-expressing these proteins as compared to the wild-type cells were analyzed at 15 °C. The analysis showed distinct sets of genes, which are possible targets of each of these proteins. This analysis will help further our understanding of the low-temperature RNA metabolism.

SUBMITTER: Phadtare S

PROVIDER: S-EPMC3456996 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Escherichia coli cold-shock gene profiles in response to over-expression/deletion of CsdA, RNase R and PNPase and relevance to low-temperature RNA metabolism.

Phadtare Sangita S

Genes to cells : devoted to molecular & cellular mechanisms 20120907 10

Cold-shock response is elicited by the transfer of exponentially growing cells from their optimum temperature to a significantly lower growth temperature and is characterized by the induction of several cold-shock proteins. These proteins, which presumably possess a variety of different activities, are critical for survival and continued growth at low temperature. One of the main consequences of cold shock is stabilization of the secondary structures in nucleic acids leading to hindrance of RNA ...[more]

PMID: 22957931

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Project description:BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Project description:BackgroundThe RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome.ResultsIn order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.ConclusionsIn this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

Dataset Information

Escherichia coli cold-shock gene profiles in response to over-expression/deletion of CsdA, RNase R and PNPase and relevance to low-temperature RNA metabolism.

Publications

Escherichia coli cold-shock gene profiles in response to over-expression/deletion of CsdA, RNase R and PNPase and relevance to low-temperature RNA metabolism.

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