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

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 develop 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. Significance Statement Together with transcription and translation, RNA decay is one of the major processes governing protein production. Here, we have developed a new statistical approach that corrects for confounding effects when estimating RNA decay rates from RNA-seq in bacteria. Our more accurate decay rate estimates indicate that bacterial transcripts have half-lives about three times shorter than previously thought. This approach allowed us to measure the effects of RNA- binding proteins (RBPs) on decay rates, identifying large cohorts of transcripts with changes in stability following RBP deletion and conditions where post-transcriptional regulation affects survival. Our method should lead to a reevaluation of RNA stability estimates across diverse bacteria and new insights into the role of RBPs in shaping the transcriptome. ### Competing Interest Statement The authors have declared no competing interest.