The interplay of RNases and sRNAs contributes to a complex layer of post-transcriptional gene regulation in proteobacteria

Abstract

Bacteria face constantly changing environmental conditions, like changes in temperature or exposure to oxidative stress. Since bacterial cells have only limited options to physically escape, a rapid adaption to the surroundings is required. As key element, gene expression is altered on transcriptional and post-transcriptional levels. This study illustrates the crucial role of polynucleotide phosphorylase (PNPase) for adaption to low temperatures, growth during organic peroxide stress, and during microaerobic growth conditions in the α-proteobacterium Rhodobacter sphaeroides. Moreover an RNA-seq analysis reavealed that the abundance of at least 334 of in total 4104 transcripts (7 %) depends on PNPase function, among them rRNAs and tRNAs which are lower abundant in a pnp mutant strain compared to the parental wild type. Additionally, PNPase influences the half-lives of different regualtory small RNAs (sRNAs) by stabilization or destabilization, among them CcsR1-4 and UpsM. Based on RNA-seq data, RNase E-, RNase III-, and PNPase-dependent differential RNA 3´ ends were globally predicted using an advanced analysis pipeline developed for this study. The results highlight a stepwise RNA processing first by endo- then by exonucleases: 5.9 % of all RNase E-dependent RNA 3´ ends and 9.7 % of all RNase III-dependent 3´ ends are further degraded by PNPase. Small RNAs are pivotal for post-transcriptional gene expression since they specificially regulate their target RNAs in various ways. During the last years, sRNAs were found to be not exclusively transcribed by an own promoter but to be derived from mRNA 5´ or 3´ untranslated regions (UTRs). So far only few examples of UTR-derived sRNAs are known from the model organisms Escherichia coli, Salmonella enterica, and R. sphaeroides. This study depicts the sRNA landscape in R. sphaeroides: According to global predictions, UTR-derived sRNAs are indeed numerous and account for 37 % of all sRNAs. In a genome wide screening five novel UTR-derived sRNAs were detected in R. sphaeroides and subsequently characterized. Some of these sRNAs, among them UdsC, were induced by oxidative stress or during different growth phases. Altered transcription rate is not always responsible for the observed changes of sRNA abundances indicating the involvement of other factors. The RNase E-dependent processing of several UTR-derived sRNAs varied for example during the early stationary growth phase and during growth under iron limiting conditions. In vivo RNase E, RNase III, and PNPase are the enzymes which are mainly involved in R. sphaeroides UTR-derived sRNA maturation, processing or degradation. These observations were strongly supported by a global predictive approach: based on multiple RNA-seq datasets obtained from different RNase mutant strains, transcription start sites and Rho-independent terminator predictions the generation mechanisms of all R. sphaeroides UTR-derived sRNA 5´ and 3´ ends were computed, emphasizing the importance of RNase E. Taken together this work provides new insights in the complex networks of post- transcriptional gene regulating in bacteria. It sheds light on the interwoven relationship between sRNAs and RNases which can either act individually or work together to form a sophisticated layer of gene regulation.