Abstract
Ribonucleases play an important role in RNA metabolism. Yet, they are also potentially destructive enzymes whose activity must be controlled. Here we describe a novel regulatory mechanism affecting RNase R, a 3' to 5' exoribonuclease able to act on essentially all RNAs including those with extensive secondary structure. Most RNase R is sequestered on ribosomes in growing cells where it is stable and participates in trans-translation. In contrast, the free form of the enzyme, which is deleterious to cells, is extremely unstable, turning over with a half-life of 2 min. RNase R binding to ribosomes is dependent on transfer-messenger RNA (tmRNA)-SmpB, nonstop mRNA, and the modified form of ribosomal protein S12. Degradation of the free form of RNase R also requires tmRNA-SmpB, but this process is independent of ribosomes, indicating two distinct roles for tmRNA-SmpB. Inhibition of RNase R binding to ribosomes leads to slower growth and a massive increase in RNA degradation. These studies indicate a previously unknown role for ribosomes in cellular homeostasis.
Highlights
Cells require mechanisms to regulate the potentially destructive action of RNases
The unusual instability of RNase R in exponential phase cells is dependent on prior binding of transfer-messenger RNA (tmRNA)-SmpB to the C-terminal region of the RNase [15]. tmRNA-SmpB binding to this region is required for recruitment of RNase R to ribosomes for its role in nonstop mRNA removal during the process of trans-translation [20]
RNase R Is Bound to Ribosomes in Exponential Phase Cells— As a first step to examine whether involvement in trans-translation might affect RNase R stability, we determined how much RNase R is bound to ribosomes
Summary
Cells require mechanisms to regulate the potentially destructive action of RNases. Results: RNase R is largely bound to ribosomes in growing cells dependent on tmRNA-SmpB and nonstop mRNA. TmRNA-SmpB binding to its C-terminal region is required for RNase R to associate with ribosomes for its participation during the process of trans-translation [19, 20]. Replacement of tmRNA with a mutant form of the RNA that still promotes proteolysis of RNase R but inhibits its binding to ribosomes results in RNase R becoming much more unstable than usual We conclude that it is the free form of RNase R that is the substrate for proteolysis and that its instability is independent of its participation in trans-translation. We find that unbound RNase R is the form of the enzyme that inappropriately degrades cellular RNAs and thereby is deleterious to cells Most importantly, these data indicate that ribosomes are able to regulate the stability and action of a deleterious bacterial protein
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