The role of endonucleases in the expression of ribonuclease II in Escherichia coli

Abstract Ribonuclease II (RNase II), encoded by the rnb gene, is one of the two major Escherichia coli exonucleases involved in mRNA degradation. Some of the ribonucleases implicated in this process have recently been shown to be inter-regulated. In this paper we studied the effects of the endonucleases RNase E and RNase III in rnb expression. We have shown that RNase E cleaves the rnb message internally: when this ribonuclease is inactivated rnb mRNA accumulates with a concomitant increase in RNase II activity. RNase III also affects RNase II expression but in an indirect way. We discuss these implications for the regulation of mRNA degradation.     

Unaltered stability of newly synthesized RNA in strains of Escherichia coli missing a ribonuclease specific for double-stranded RNA

Pairs of very closely related Escherichia coli strains were prepared, one having the wild-type allele for ribonuclease III, an enzyme which specifically degrades double-stranded RNA, and the other having a mutant RNase III allele. Growth and phage plating efficiency were compared in these strains. The RNase III+ strains grow better than the RNase III- strains and plate T7 and lambda phage better, but T4 plates with the same efficiency on both strains. On the other hand, the half lives of newly synthesized RNA as well as of functional beta-galactosidase mRNA are similar in both kind of strains. These two parameters, however, are significantly longer in both strains as compared to the original strain from which they were derived. Also, no difference in the differential induction of beta-galactosidase was observed between such strains. Thus, we have to conclude that either ribonuclease III does not play a significant role in the functioning and stability of newly synthesized mRNA, or that enough enzymatic activity was left, residual RNase III or some other enzyme to deal with double-stranded regions in the message.     

RNase III controls the degradation of corA mRNA in Escherichia coli

In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.     

RNase III-mediated silencing of a glucose-dependent repressor in yeast

Members of the RNase III family are found in all species examined with the exception of archaebacteria, where the functions of RNase III are carried out by the bulge-helix-bulge nuclease (BHB). In bacteria, RNase III contributes to the processing of many noncoding RNAs and directly cleaves several cellular and phage mRNAs. In eukaryotes, orthologs of RNase III participate in the biogenesis of many miRNAs and siRNAs, and this biogenesis initiates the degradation or translational repression of several mRNAs. However, the capacity of eukaryotic RNase IIIs to regulate gene expression by directly cleaving within the coding sequence of mRNAs remains speculative. Here we show that Rnt1p, a member of the RNase III family, selectively inhibits gene expression in baker's yeast by directly cleaving a stem-loop structure within the mRNA coding sequence. Analysis of mRNA expression upon the deletion of Rnt1p revealed an upregulation of the glucose-dependent repressor Mig2p. Mig2p mRNA became more stable upon the deletion of Rnt1p and resisted glucose-dependent degradation. In vitro, Rnt1p cleaved Mig2p mRNA and a silent mutation that disrupts Rnt1p signals blocked Mig2p mRNA degradation. These observations reveal a new RNase III-dependent mechanism of eukaryotic mRNA degradation.