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Improved RNA stability estimation through Bayesian modeling reveals most bacterial transcripts have sub-minute half-lives [CLIP-seq]

ABSTRACT: RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in post-transcriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 minute, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with CLIP-seq revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3' untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for both proteins in the control of respiration, and for ProQ in the oxidative stress response. Our findings provide new insights into post-transcriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium

PROVIDER: GSE234007 | GEO | 2024/03/26

REPOSITORIES: GEO

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Project description:RNA decay was measured in Prochlorococcus after inhibition of transcription by rifampicin using customized Affymetrix gene expression arrays. RNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans. Using a combination of microarrays, quantitative RT-PCR and a new algorithm for determining RNA decay rates, we found a median half-life of 2.4 min and a median decay rate of 2.6 min for expressed genes â two-fold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (ca. 18 min) were for highly expressed genes. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb. We hypothesize that the fast turnover relative to generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will inform on the modelling of cell processes and help interpret the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms. Prochlorococcus cells were treated with rifampicin, which prevents initiation of new transcripts. Cells were harvested at 0 min (before rifampicin addition), 2.5 min, 5 min, 10 min, 20 min, 40 min and 60 min after rifampicin addition.