Bacillus subtilis mRNA metabolism and gene expression : a study on the function and structure of the novel and essential ribonucleases Y and J – RNAJAY

In order to identify potential RNase Y substrates in the cell we want to measure the mRNA levels in a wild type strain and compare them to a strain where RNase Y has been depleted. A priori, RNAs cleaved by RNase Y should be more abundant in the depletion strain. Using array transcriptome technology will permit to analyze all transcripts present in the bacteria at the same time.

The first significant results that we have obtained are the identification of hundreds of new RNase Y substrates revealed by a transcriptome analysis. This study is under evaluation and will allow to conclude on the importance of RNase Y in the initiation of degradation of all RNA present in the cell.
A partial proteolysis analysis of RNase Y has revealed the presence of a C-terminal domain which is more stable than the entire protein and might be useful for the generation of cristals for X-ray structure determination.

It is already quite safe to say that RNase Y is a key nuclease which determines the stability of hundreds or even thousands of transcripts in Bacillus subtilis. Understanding the structural and biochemical aspects of RNase Y function of RNase Y but also RNase J should allow to envisage them as targets for elaborating novel antibiotiques. Indeed, a very large number of bacterial species including many pathogens contain at least a RNase J or Y.

The evaluation of the role of RNase Y on the expression of all RNA expressed in Bacillus subtilis is underway and an associated publication is in preparation.

The processing and degradation of mRNA is a key element in the control of procaryotic gene expression. In Escherichia coli, RNase E has a pivotal role in these processes but Bacillus subtilis and other Gram positive bacteria have a very different set of enzymes directing mRNA metabolism and the major players have emerged only very recently. Indeed, B. subtilis has the essential RNase J1 and its paralog RNase J2, dual activity enzymes that have an RNase E-like endoribonucleolytic and a 5’-3’ exoribonucleolytic activity that requires a monophosphorylated 5’ end. The 3D structure of RNase J has revealed a surprising conservation of the overall 3D architecture between RNase J and RNase E. This constitutes an interesting case of convergent evolution whose significance is not yet understood. Group 1 will analyse the significance of this conservation by creating chimeric proteins between RNase E and RNase J1.
In theory, the polyvalence of RNases J1/J2 could explain all characteristics described for mRNA degradation in B. subtilis, including a decay-initiating endonucleolytic cleavage followed by 5’ exonucleolytic degradation. However, inactivation/depletion of RNases J1/J2 only slightly increases global mRNA half-life, an observation which is not in favor of an important function for these nucleases in initiating global mRNA decay.

Recently, group 1 has characterized a novel and essential endoribonuclease (RNase Y) in B. subtilis. Despite an absence of sequence similarity, RNase Y shares extensive functional homologies with RNase E ; e.g. the activity of both enzymes is stimulated by a monophosphorylated 5’-end of the substrate and RNase Y is the only nuclease known, besides RNase E, to have a significant effect on global mRNA half-life.

This raises the fundamental question on the respective contributions of 5’-3’ exonucleolytic and endonucleolytic cleavage in mRNA decay in Gram positive organisms that do not contain RNase E. Group 1 proposes to study the role of RNases Y and J1/J2 in the initiation of mRNA decay and the control of gene expression. They will also analyse the parameters that determine the specificity and cleavage site recognition of RNase Y. Both a global approach (e.g. tiling array study) and a detailed analysis of individual substrates by in vitro and in vivo techniques will be used.
The principal aim of this study is to confirm or invalidate the notion that mRNA processing and degradation might be more similar between B. subtilis and E. coli than presently assumed, with an endonucleolytic cleavage being the crucial step in initiating mRNA decay in both organisms. In this scenario, RNase J would be mostly involved in degrading 3’ cleavage products exonucleolytically in a 5’-3’ direction. This property may have evolved in organisms like B. subtilis that, in contrast to E. coli, does not appear to have an efficient polyadenylation pathway to allow 3’-5’ degradation of structured RNA (e.g. containing a terminator at the 3’ end).
In parallel, group 2 will attempt to determine the crystal structure of RNase Y, alone and in the presence of RNA. The initial in vitro characterization of RNase Y performed by group 1 will be followed up by group 2 using a more quantitative approach including biophysical methods that will allow to determine important parameters related to RNase Y activity including catalytic constants, quaternary structure, protein dynamics, etc.

RNases Y and J probably represent the key enzymes directing RNA metabolism in B. subtilis and numerous other Gram positive species including many pathogens like Listeria, Staphylococcus aureus, Streptococcus or B. anthracis. The study of these two essential ribonucleases should produce important fundamental knowledge on mRNA metabolism/gene regulation that will concern many other eubacterial species where these enzymes are conserved. In addition, the often essential nature of these RNases make them potential targets for the design of new antimicrobial drugs.