Kinetic model of small RNA-mediated regulation suggests that a small RNA can regulate co-transcriptionally
Matthew A. ReyerShriram ChennakesavaluEmily M. HeidemanXiangqian MaMagda BujnowskaLu HongAaron R. DinnerCarin K. VanderpoolJingyi Fei
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Abstract Small RNAs (sRNAs) are important regulators of gene expression in bacteria, particularly during stress responses. Many genetically and biochemically well characterized sRNAs regulate gene expression post-transcriptionally, by affecting translation and degradation of the target mRNA after they bind to their targets through base pairing. However, how regulation at each of these levels quantitatively contributes to the overall efficacy of sRNA-mediated regulation is not well understood. Here we present a general approach combining imaging and mathematical modeling to determine kinetic parameters at different levels of sRNA-mediated gene regulation. Unexpectedly, our data reveal that certain previously characterized sRNAs are able to regulate some targets co-transcriptionally, rather strictly post-transcriptionally, and suggest that sRNA-mediated regulation can occur early in the mRNA’s lifetime, perhaps as soon as the sRNA binding site is transcribed. In addition, our data suggest several important kinetic steps that may determine the efficiency and differential regulation of multiple mRNA targets by an sRNA. Particularly, binding of sRNA to the target mRNA is likely the rate-limiting step and may dictate the regulation hierarchy observed within an sRNA regulon.Keywords:
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Bacterial pathogens regulate virulence factor gene expression coordinately in response to environmental stimuli, including nutrient starvation. The phosphate (Pho) regulon plays a key role in phosphate homeostasis. It is controlled by the PhoR/PhoB two-component regulatory system. PhoR is an integral membrane signaling histidine kinase that, through an interaction with the ABC-type phosphate-specific transport (Pst) system and a protein called PhoU, somehow senses environmental inorganic phosphate (P(i)) levels. Under conditions of P(i) limitation (or in the absence of a Pst component or PhoU), PhoR activates its partner response regulator PhoB by phosphorylation, which, in turn, up- or down-regulates target genes. Single-cell profiling of PhoB activation has shown recently that Pho regulon gene expression exhibits a stochastic, "all-or-none" behavior. Recent studies have also shown that the Pho regulon plays a role in the virulence of several bacteria. Here, we present a comprehensive overview of the role of the Pho regulon in bacterial virulence. The Pho regulon is clearly not a simple regulatory circuit for controlling phosphate homeostasis; it is part of a complex network important for both bacterial virulence and stress response.
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The expression of a vegetative catalase gene, katA (formerly the kat-19 gene), is necessary to protect Bacillus subtilis from H2O2, presumably by removing the oxidant from the environment. Genetic analysis of katA revealed that this gene is under two distinct forms of regulation, temporal and H2O2 inducible. The results reported here demonstrate that (i) the H2O2-inducible regulation of katA gene is not a component of the SOS regulon, (ii) the regulatory genes spo0A and abrB are involved in the temporal regulation but not the H2O2-specific induction of katA gene expression, and (iii) transcription initiation for the katA gene occurs at the same site under both forms of regulation.
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Several research advances have indicated an important role of transfer RNA (tRNA)-derived small RNAs in modulating developmental processes or stress responses. Recently, from the deep sequencing of small RNAs in Arabidopsis (Arabidopsis thaliana), we identified a new class of 19-nucleotide (nt) small RNAs corresponding to the 5′ end of tRNA accumulated at high levels in phosphate-starved roots. In two very recent studies, 19-nt tRNA fragments were also observed in human cells, suggesting their widespread nature. In our study, tRNA halves cleaved at the anticodon loop, the most common tRNA fragments found, were predominant in roots. These results showed a spatial and temporal expression pattern of small RNAs derived from specific cleavage of tRNA molecules. Although the function of these tRNA-derived small RNAs under phosphate deficiency remains unknown, their diversity, biogenesis and potential function are henceforth summarized and discussed. Certainly, they will emerge as a novel class of regulatory small RNAs.
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Abstract Cells cope with and adapt to ever-changing environmental conditions. Sophisticated regulatory networks allow cells to adjust to these fluctuating environments. One such archetypal system is the S. cerevisiae Pho regulon. When external inorganic phosphate ( P i ) concentration is low, the Pho regulon activates, expressing genes that scavenge external and internal P i . However, the precise mechanism controlling this regulon remains elusive. We conducted a systems analysis of the Pho regulon on the single cell level under well-controlled environmental conditions. This analysis identified a robust, perfectly adapted Pho regulon state in intermediate P i conditions, and we discovered a hitherto unknown intermediate nuclear localization state of the transcriptional master regulator Pho4p. The existence of an intermediate nuclear Pho4p state unifies and resolves outstanding incongruities associated with the Pho regulon, explains the observed programmatic states of the Pho regulon, and improves our general understanding of how nature evolves and controls sophisticated gene regulatory networks. We further propose that robustness and perfect adaptation are not achieved through complex network-centric control, but by simple transport biophysics. The ubiquity of multi-transporter systems suggests that similar mechanisms could govern the function of other regulatory networks as well.
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Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5´ nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that captures a more complete picture of sRNA. By tagging 5´P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5ʹ-terminal modifications (5´X), with a selective approach for 5-phosphorylated sRNA (5´P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5´ insensitive library strategies with 5´ sensitive tagging, we have successfully tackled an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.
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Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS- bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.
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