Ribosomes regulate the stability and action of the exoribonuclease RNase R

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.

Keywords: Escherichia coli; Protein Stability; RNA Metabolism; Ribonuclease; Ribosomes.

FIGURE 1.

RNase R binds to ribosomes. A, amount of ribosome bound and free RNase R in exponential (Exp) and stationary (Sta) phase wild type cells. The amount of RNase R in each fraction was determined by Western blotting. Note that a 5-fold higher amount of soluble protein was added for exponential phase cells than for stationary phase cells. B, amount of ribosome- bound and -free RNase R in exponential phase WT, C-terminal truncated (ΔC), and N-terminal truncated (ΔN) RNase R mutant strains. The data shown are the average of three independent experiments. The amount of ribosome bound RNase R in wild type cells was set at 1. Error bars indicate the S.E. C, quantification of tmRNA and RNase R in exponential phase cells. Measurement of tmRNA and RNase R (RNR) concentration by Northern blotting and Western blotting was carried out as described under “Experimental Procedures.” The data shown are from a representative experiment carried out a minimum of two times. The values for exponential phase tmRNA and RNR (Exp) were determined by the intensity of their respective bands relative to those of the standards and are shown in italics.

FIGURE 2.

Overexpression of nonstop mRNA increases RNase R binding to ribosomes. A, total amount of RNase R (RNR), FLAG-SmpB, and tmRNA in cells overexpressing stop or nonstop c1 mRNA for 10 min determined by Western or Northern analysis. B, percent of RNase R binding to ribosomes in wild type cells overexpressing stop or nonstop c1 mRNA determined by Western blotting. The average of three independent experiments is shown. Error bars indicate the S.E. C, percent of ribosome-bound and free RNase R in smpB mutant cells overexpressing nonstop c1 mRNA determined by Western blotting. Average of three independent experiments is shown. Error bars indicate the S.E. D, amount of rnr and smpB mRNAs in cells overexpressing nonstop c1 mRNA. The culture was induced by arabinose for the indicated times, and total RNA was extracted. After reverse transcription, partial sequences of rnr and smpB were amplified with gene-specific primers, and the PCR products were detected by ethidium bromide staining. E, half-life of RNase R in cells overexpressing nonstop c1 mRNA. The time after nonstop mRNA induction is shown on the right, and the time after chloramphenicol (CM) addition is shown at the top. ind, induction.

FIGURE 3.

Ribosome binding stabilizes RNase R. A, amount of total tmRNA, ribosome-bound tmRNA, and RNase R (RNR) in WT and a tmRNA mutant (Mut) strain. B, degradation of RNase R in vitro by HslUV protease in the presence of WT or Mut tmRNA. C, amount of RNase R binding to ribosomes in WT and tmRNA Mut strains. The data shown are the average of three independent experiments. The amount of ribosome bound RNase R in wild type cells was set at 1. Error bars indicate the S.E. D, half-life of full-length RNase R (RNR-FL) in WT and tmRNA Mut strains. The numbers below the lanes are the amount of RNase R remaining at each time point. CM, chloramphenicol. E, half-life of full-length RNase R (RNR-FL) in hslv/lon and hslv/lon/tmRNA Mut strains. F, half-life of C-terminal-truncated RNase R (RNR-ΔC) in WT and tmRNA Mut strains. G, half-life of N-terminal truncated RNase R (RNR-ΔN) in WT and tmRNA Mut strains. In all experiments RNase R was determined by Western blotting.

FIGURE 4.

Effect of ribosomes on RNase R degradation in vitro. A, proteolysis of RNase R by HslUV protease in the presence of ribosomes containing stop or nonstop mRNA carried out as described under “Experimental Procedures.”. B, proteolysis of RNase R by HslUV protease in the presence of tmRNA or tmRNA charged with Ala in the presence or absence of nonstop ribosomes. RNase R remaining was detected by Western blotting with RNase R-specific antibody.

FIGURE 5.

Effect of binding of catalytically inactive RNase R to ribosomes. A, amount of RNase R (RNR) in WT and D278N mutant cells determined by Western blotting. B, half-life of RNase R in wild type and D278N mutant cells. Times after chloramphenicol (CM) addition are shown at the top. C, amount of RNase R binding to ribosomes in WT and D278N strains. The data shown are the average of three independent experiments. The amount of ribosome bound RNase R in wild type cells was set at 1. Error bars indicate the S.E. D, doubling time of WT and D278N strains determined by competition experiments.

FIGURE 6.

Effect of a rimO mutation on RNase R binding to ribosomes. A, amount of RNase R (RNR) in WT and rimO mutant cells determined by Western blotting. B, half-life of RNase R in wild type and rimO mutant cells. Time after chloramphenicol (CM) addition is shown at the top. C, amount of RNase R binding to ribosomes in WT and rimO strains. The data shown are the average of three independent experiments. The amount of ribosome bound RNase R in wild type cells was set at 1. Error bars indicate the S.E.

FIGURE 7.

Effect of elevated free RNase R on growing cells. A, doubling time of WT, K544R, and ΔC mutant strains determined by competition experiments. B, amount of acid-soluble radioactivity present in WT, K544R, and ΔC mutant strains. Degradation of RNAs was determined from the release of acid-soluble radioactivity presented as a percentage of the total radioactive RNA in the culture sample. The data shown are the average and S.D. based on three independent experiments.