Regulation of RNA maturation and turnover by antisense RNA – asSUPYCO

We have chosen to study a restricted number of specific examples of small RNAs from among the large number of recently discovered sRNAs in E. coli, B. subtilis and H. pylori, that will serve as models. To study their precise function in vivo, we will use genetic approaches: mutations that either inactivate or overproduce the sRNA will be introduced into the host bacteria. The effect of this inactivation or overproduction of sRNAs on their targets will be determined using molecular biology techniques that allow us to measure both the quality and the quantity of RNAs. The regulatory mechanism of many sRNAs relies on their ability to modulate the degradation of their targets by ribonucleases. The three bacterial species studied in this project possess different degradation machineries. To fully understand their roles in regulation by sRNAs, we will use mutant bacterial strains lacking some of the key RNases. New high throughput sequencing techniques will be used to determine their contribution at the whole-genome level. Lastly, we will attempt to reconstitute some of these regulatory events in vitro, to better understand the mechanisms involved and the contribution of each partner in the best detail possible.

We have confirmed the expression of a certain number of small antisense RNAs in E. coli, B. subtilis and H. pylori. We have identified the RNases involved in their degradation and, for some, determined their function and mechanism of action. In Bacillus subtilis, for example, we have shown that two small RNAs are involved in the inhibition of the expression of mRNAs encoding toxic peptides. In both cases the small RNA and mRNA base-pair and the hybrid is degraded by RNase III, an ribonuclease with specificity for double-stranded helices. Thanks to this regulatory mechanism, the bacteria can survive despite the presence of these genes, as the toxins encoded by these mRNAs are not produced. This study explains the essential function of RNase III in B. subtilis, whereas it is dispensable in a large number of bacteria.

The study showing that RNase III is necessary to silence mRNAs encoding toxins in B. subtilis may also apply to other essential ribonucleases in other organisms.
The transcriptome analysis of bacterial strains lacking certain RNases or RNase co-factors has allowed us to identify new potential regulatory RNAs. One of the goals of this work is thus to determine whether some of these new RNAs have regulatory function and what is their mechanism of action?

Articles :
- Durand, S., Gilet, L. and Condon, C. (2012) The essential function of B. subtilis RNase III is to silence foreign toxin genes. PLoS Genet, 8(12): e1003181. doi: 10.1371/journal.pgen.1003181
- A. Maes, C. Gracia, D. Bréchemier, P. Hamman, E. Chatre, L. Lemelle, P. N. Bertin & E. Hajnsdorf (2013) “Role of polyadenylation in regulation of the flagella cascade and motility in E. coli“ Biochimie, 95, 410-418.
- P. Régnier and E. Hajnsdorf (2013) «The interplay of Hfq, poly(A) polymerase I and exoribonucleases at the 3' ends of RNAs resulting from Rho-independent termination: a tentative model« RNA Biology 10(4).
- P. Boudry, C. Gracia, M. Monot, J. Caillet, L. Saujet, E. Hajnsdorf, B. Dupuy, I. Martin-Verstraete & O. Soutourina (2014) «Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile« J. Bacteriol sous presse.
- J. Caillet J., C. Gracia C., F. Fontaine and E. Hajnsdorf (2014) «Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function« RNA sous presse
- Iost, I., Bizebard, T. and Dreyfus, M. (2013) Functions of DEAD-box proteins in bacteria: Current knowledge andpending questions. Biochim Biophys Acta, 1829, 866-877.

The study of regulation of gene expression by small RNA molecules (sRNA) has seen an exponential growth in interest and intensity over the past 10 years. The vast majority of these studies focus on the effect of trans-encoded sRNAs on the translation of their target genes. However, some recent studies have shown that direct control of mRNA stability is an alternative method of regulating gene expression by sRNAs. In this project we propose to focus primarily, but not exclusively, on cis-encoded antisense RNAs (asRNA) and to determine whether and how they control the maturation and/or stability of their sense strand targets. These studies will be performed in three evolutionarily distant organisms with different RNA degradation machineries: the Gram-positive and Gram-negative model organisms, Bacillus subtilis and Escherichia coli, each with an extensive array of ribonucleases, and the human pathogen Helicobacter pylori, which has a minimal set of RNA processing and degradation enzymes. There has been a huge influx of data predicting new regulatory RNAs in these organisms in recent times and mechanistic studies have unable to keep up with the pace. We therefore propose to study specific examples of the regulation by asRNA in each of the three species. We will study asRNAs complementary to mRNAs encoding the PNPase ribonuclease, ribosome-associated proteins and toxic peptides. Moreover, a new class of asRNA complementary to stable RNA (rRNA and tRNA) has been identified and their role in rRNA/tRNA maturation/degradation and ribosome biogenesis will be investigated. For all these asRNA examples, we propose to determine their mechanisms of action and the RNases involved. Finally, we will use mutants in RNases or accessory proteins such as poly(A) polymerase to identify their roles in general regulatory RNA metabolism at a genome-wide level.