RNA MATURATION/DEGRADATION IN MYCOBACTERIA: IN VIVO AND IN VITRO CHARACTERIZATION OF RNASE J AND RNASE E

Escherichia coli and Bacillus subtilis have very different sets of ribonucleases, in particular the presence of RNase E and RNase J, respectively, that have been used to explain significant differences in RNA metabolism between these the two model organisms. However, these studies might have somewhat polarized our view of RNA metabolism, while recent works outline models of RNA degradation that are more similar than has been thought. In fact, the recent characterization of RNase Y as the scaffold for the degradosome assembly in B. subtilis lead to the consideration that RNA degradation in B. subtilis might begin through an endonucleolitycal cleavage, followed by exonucleolytical degradation. In this work, we have identified a functional RNase J in Mycobacterium smegmatis and characterized its in vitro 5’-3’ exo- and endonucleolytic activities. Furthermore, we constructed two mutants in M. smegmatis rnj: a conditional and a knock out mutant, thus demonstrating that in M. smegmatis the gene is not essential, contrary to the RNase J1 function in B. subtilis. In M. smegmatis RNase J co-exists with RNase E, a configuration that enabled us to study how these two key nucleases collaborate. A conditional mutant in the rne gene was constructed, demonstrating that this function is essential for M. smegmatis, as it is in E. coli. Moreover, a conditional mutant in Mycobacterium tuberculosis, confirmed its essentiality also in this organism. We studied the respective roles of the M. smegmatis RNase J and RNAse E ribonucleases in the 5’ end maturation of the katG transcript, previously demonstrated to derive from an endoribonucleolytic processing. Here we find that RNase E is responsible of the specific cleavage of the 5’ katG end. Further, we show that RNase E and RNase J are involved in the 5’ end processing of all three ribosomal RNAs. Thus the maturation pathways of rRNAs in M. smegmatis are quite different from those observed in both E. coli and B. subtilis. Studying organisms containing different combinations of key ribonucleases can thus significantly broaden our view of the strategies directing RNA metabolism used by various organisms.