RNA polyadenylation and cellular ATP levels regulate the stability of RNAs in Escherichia coli
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ABSTRACT:
PROVIDER: PRJNA1044098 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26923656.fastq.gz | Fastqsanger.gz | |||
| SRR26923657.fastq.gz | Fastqsanger.gz | |||
| SRR26923658.fastq.gz | Fastqsanger.gz | |||
| SRR26923659.fastq.gz | Fastqsanger.gz | |||
| SRR26923660.fastq.gz | Fastqsanger.gz |
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Project description:To investigate the extent of the effect of poly(A) polymerase (PAP I)-mediated polyadenylation on RNA stability, we performed the first genome-wide study of RNA stability in the absence of PAP I activity. Inactivation of the pcnB gene coding for PAP I led to a global stabilization of E. coli RNAs, with 1403 stabilized transcripts and only 4 destabilized. Stabilized RNAs were involved in essential cellular functions such as DNA replication and repair, translation, RNA degradation, central carbon metabolism but also in stress responses. Because PAP I is an ATP-consuming enzyme we wondered whether the RNA stabilization observed after inactivation of PAP I could also be related to changes in intracellular ATP levels. We demonstrated for the first time in E. coli, that lowering intracellular ATP levels below 1 µM/OD stabilizes RNAs. Although the ATP level was reduced by 20 % in the pcnB mutant on glucose, the ATP level was still too high to have any role in the observed RNA stabilization. However, in experiments where the ATP level was artificially strongly decreased, inactivation of PAP I by substrate availability was implicated in the stabilization mechanism of certain RNAs. This study clearly demonstrates that PAP I is at the crossroads of the regulation of RNA stability by energy status in E. coli cells.
2024-11-27 | GSE248472 | GEO
Project description:The post-transcriptional process of RNA polyadenylation sits at the crossroads of energy metabolism and RNA metabolism. RNA polyadenylation is catalyzed by poly(A) polymerases which use ATP as a substrate to add adenine residues to the 3’ end of RNAs, which can alter their stability. In E. coli, RNA polyadenylation mediated by the major poly(A) polymerase (PAP I) was previously shown to facilitate degradation of individual RNAs. In this study, we performed the first ever genome-wide study of RNA stability in the absence of PAP I. Inactivation of the pcnB gene coding for PAP I led to the stabilization of more than a thousand of E. coli RNAs in the form of full-length functional molecules or non-functional fragments, involved in essential cellular functions such as DNA replication and repair, translation, central carbon metabolism, and stress response. The absence of PAP I also altered the energy metabolism, with an almost 20 % reduction in ATP levels. To better understand how RNA and energy metabolisms are interconnected, we investigated the role of ATP levels in regulating RNA stability. When we lowered intracellular ATP levels below 0.5 mM, certain RNAs were stabilized demonstrating the causal link between ATP levels and RNA stability for the first time in E. coli. Above this concentration, changes in ATP levels had no impact on RNA stability. We also demonstrated that some RNAs were stabilized when PAP I was inactivated by low ATP availability. These results clearly demonstrate that PAP I mediates an energy-dependent RNA stabilization which may contribute to cell energy homeostasis under energy-limited conditions.
2024-11-27 | GSE272651 | GEO
Project description:RNA stability is regulated by both RNA polyadenylation and ATP levels, linking RNA and energy metabolisms in Escherichia coli
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Project description:mRNA stability plays an important role in gene expression. Here, using 3’ end sequencing of newly made and steady-state poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having high GC content and likely more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to other two. These features also differentiate alternative polyadenylation (APA) 3’UTR isoforms in their stability difference in a cell type-specific manner. By analyzing cell differentiation of a neural stem cell line, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirms the neuronal identity of SH-SY5Y cells at gene expression and APA levels. Through comparison of APA isoforms, we show that mRNA stability control buffer the cleavage and polyadenylation site (PAS) choice in the cells studied. In addition, transcripts using intronic PAS are generally less stable, especially when the intron harboring the PAS is large and has a strong 5’ splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Moreover, we found that poly(A)+ RNAs encoded by the mitochondrial genome are more stable in SH-SY5Y cells than the other two cell types, suggesting that mitochondrial RNA metabolism is distinct in neurons. Together, our results reveal multiple cell-specific mechanisms by which mRNA stability could contribute to the gene expression and APA programs that are important for cell identity.
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Project description:Cells have compensatory mechanisms to coordinate the rates of major biological processes, thereby permitting growth in a wide variety of conditions. Here, we uncover a compensatory link between cleavage/polyadenylation in the nucleus and mRNA turnover in the cytoplasm. On a global basis, same-gene 3’ mRNA isoforms with > two-fold differences in half-lives have steady-state mRNA levels that differ by significantly less than a factor of two. In addition, increased efficiency of cleavage/polyadenylation at a specific site is associated with reduced stability of the corresponding 3’ mRNA isoform. This inverse relationship between cleavage/polyadenylation and mRNA isoform half-life reduces the variability in the steady-state levels of mRNA isoforms, and it occurs in all four growth conditions tested. These observations suggest that during cleavage/polyadenylation in the nucleus, mRNA isoforms are marked in a manner that persists upon translocation to the cytoplasm and affects the activity of mRNA degradation machinery, thus influencing mRNA stability.
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Project description:Pseudouridylation is coupled with polyadenylation ot promote mRNA stability
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Project description:Alternative polyadenylation isoform abundance and stability of HEK293T cells
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Project description:Here, we developed a Ψ RNA immunoprecipitation sequencing method (Ψ-RIP-seq) combined with direct RNA sequencing by Oxford Nanopore Technologies to detect mRNA Ψ transcriptome-wide. We identified a large number of novel Ψs in mRNA, revealing a conserved enrichment of Ψ in mRNA 3′ UTR. We further showed that a genome-wide association of Ψ with mRNA polyadenylation, and uncovered that cleavage factor complex I (CFI) regulated Ψ generation in mRNA 3′ UTR. We finally demonstrated that Ψ in mRNA 3′ UTR promoted mRNA stability in a CFI-dependent manner.
2024-09-15 | GSE184349 | GEO
Project description:Here we demonstrate a new regulatory mechanism for tRNA processing in Escherichia coli whereby RNase T and RNase PH, the two primary 3' → 5' exonucleases involved in the final step of 3'-end maturation, compete with poly(A) polymerase I (PAP I) for tRNA precursors in wild-type cells. In the absence of both RNase T and RNase PH, there is a >30-fold increase of PAP I-dependent poly(A) tails that are ≤10 nt in length coupled with a 2.3- to 4.2-fold decrease in the level of aminoacylated tRNAs and a >2-fold decrease in growth rate. Only 7 out of 86 tRNAs are not regulated by this mechanism and are also not substrates for RNase T, RNase PH or PAP I. Surprisingly, neither PNPase nor RNase II has any effect on tRNA poly(A) tail length. Our data suggest that the polyadenylation of tRNAs by PAP I likely proceeds in a distributive fashion unlike what is observed with mRNAs.
| S-EPMC3378859 | biostudies-literature
Project description:mRNA degradation is a critical aspect of gene expression which dictates mRNA steady-state levels in conjunction with transcription rate. Previous studies in other model organisms have demonstrated that enrichment of specific codons within the open reading frame (ORF) can influence mRNA stability, primarily by modulating translation elongation speed. Despite advancements in our understanding of mRNA stability regulation by microRNAs and 3'UTR-based factors in mammalian systems, the importance of other mRNA regions such as the ORF sequence on dictating cellular mRNA levels are incompletely understood. To characterize the effects of the coding sequence on mRNA decay in mammals, we analyzed mRNA stability in human and Chinese Hamster Ovary (Cricetulus griseus) cells by both global metabolic labeling and single-gene mRNA reporter transcription shutoffs. In agreement with previous studies, we observed that synonymous codon usage impacts mRNA stability in mammalian cells. Unexpectedly, we also found that amino acid content is a potent determinant of mRNA stability in humans and other mammalian species. Codon and amino acid effects on decay correlate with tRNA levels measured by tRNA-Seq or intracellular amino acid levels measured by HPLC, respectively. These results suggest that both tRNA and amino acid levels have complementary effects on regulation of mRNA stability in mammals, hinting at the possibility of dynamic control of mRNA levels via altered tRNA or amino acid levels.
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