Regulation of RNase R by ribosomes

Ribonucleases play an important role in RNA metabolism. Yet, they are also potentially destructive enzymes whose activity must be controlled. We describe here 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 tmRNA-SmpB, non-stop 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. INTRODUCTION Ribonucleases (RNases) are important participants in essentially every aspect of RNA metabolism (1–5). Yet, they are also destructive enzymes that potentially could cause serious problems with a cell’s complement of RNA. As a consequence, we would expect that cells have evolved mechanisms to protect RNA against such possible dangers, although, at present, very little information is available in this area. As possible general mechanisms, one can envision strategies in which the RNA is protected by its structure, http://www.jbc.org/cgi/doi/10.1074/jbc.M113.519553 The latest version is at JBC Papers in Press. Published on October 16, 2013 as Manuscript M113.519553 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 0, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of RNase R by ribosomes 2 sequestration, or location in the cell. Alternatively, the RNases themselves could have evolved to be highly specific for a limited number of substrates or they could be subject to strict regulation. In a cell such as Escherichia coli, with at least twenty RNases, it would not be surprising if multiple mechanisms come into play. Our laboratory has been exploring one highly unusual regulatory process that affects the 3' to 5' exoribonuclease, RNase R (6, 7). Since RNase R is processive and is able to digest all RNAs, even those with extensive secondary structure (8–12), it has the potential to be an extremely destructive enzyme. In fact, overexpression of RNase R is known to be deleterious to cells (7). Hence, it is important that the activity of RNase R be carefully controlled, and this is accomplished by regulating the protein’s stability. RNase R is very unstable in growing E. coli, but is stable in stationary phase cells (13, 14). Its instability is determined by the acetylation of a single lysine residue, Lys544, which stimulates binding of the trans-translation factors, tmRNA and SmpB, to the C-terminal region of the RNase (15, 16). The binding of tmRNA-SmpB stabilizes association of the proteases, HslUV or Lon, with the N-terminal region of RNase R resulting in proteolysis (17). Since stationary phase RNase R is not acetylated due to the absence of the acetylating enzyme (18), tmRNA and SmpB bind poorly, and, as a result, RNase R is completely stable (16). tmRNA-SmpB binding to its C-terminal region is also required for RNase R to associate with ribosomes for its participation during the process of trans-translation (19, 20). Thus, it was of considerable importance to determine whether RNase R instability might be a consequence of its role in trans-translation, and to determine what role ribosomes might play in the instability of RNase R. We show here that RNase R is largely bound to ribosomes in growing cells, and that it is dependent on the amount of non-stop mRNA. Interestingly, this binding actually stabilizes RNase R. 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 also 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. EXPERIMENTAL PROCEDURES Materials—Antibody against RNase R was prepared and purified as described previously (7, 15). Anti-FLAG M2 mAb was from Sigma. Anti-rabbit and anti-mouse IgG HRP conjugate were obtained from Santa Cruz Biotechnology. [γ-P]ATP, L-[C] alanine and [H]-uridine were purchased from PerkinElmer Life Sciences and GE Healthcare, respectively. RNeasy mini kit was from Qiagen. Protease inhibitor cocktail was purchased from Calbiochem. Ni-NTA His-bind resin was obtained from Novagen. M-MLV reverse transcriptase and RNasin were from Promega. Purification of tmRNA, SmpB and RNase R was described previously (15, 16). All other materials were reagent grade. Bacterial Strains and Growth Conditions—All strains used were derivatives of E. coli K12 strain MG1655(Seq)rph and were previously described (15–18). The rimO insertion mutant allele was provided by Dr. Michael Brad Strader, U.S. Food and Drug Administration (21). The rimO and rna (encoding RNase I) mutant genes were each by gest on O cber 0, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of RNase R by ribosomes 3 introduced into wild type or indicated mutant strains by phage P1-mediated transduction using P1vir. Site-directed mutagenesis of tmRNA G3 to A in the chromosome was performed with oligo T1 (Table 1), using a previously described protocol (15). Aspartic acid 278 of RNase R was changed to asparagine with oligo R1 (Table 1). Recombinants were selected by PCR with primers T2 and T3, or R2 and R3 (Table 1), respectively, and the resulting gene mutations were confirmed by DNA sequencing (22). Cells were grown at 37 °C in liquid culture in YT medium or M9-glucose medium. Antibiotics, when present, were at the following concentrations: kanamycin, 50 μg/ml; ampicillin, 100 μg/ml; chloramphenicol, 34 μg/ml. Exponential phase cells were collected at an A550 of ~0.3, and cells grown overnight were used as stationary phase samples. Ribosome Isolation — Exponential and stationary phase wild type or indicated mutant strain cells were disrupted in lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NH4Cl, 20 mM MgCl2, 2 mM β-ME and 1mg/ml DNase I) containing protease inhibitor cocktail as described (15). The suspension was centrifuged at 30,000g for 10 min to remove cell debris. The supernatant fraction was further centrifuged at 100,000g for 90 min and the resulting supernatant and pellet fractions were used to detect the amount of unbound and ribosome-bound RNase R or tmRNA, respectively (23). RNase R and tmRNA Cellular Concentration Measurement—Wild type cells were grown to an A550 of ~0.3 and were collected by centrifugation. The concentration of RNase R and tmRNA were determined as previously described (24, 25) using Western blotting and Northern blotting, respectively. Induction of c1 mRNA—The coding sequence for the N-terminal domain of the bacteriophage c1 gene followed by the strong trp operon transcriptional terminator (26) was amplified by PCR with primers C1 and C2 (Table 1) using λ DNA as template. The PCR product was purified and digested with NcoI and HindIII, and then cloned into the corresponding sites on pBAD24 (non-stop mRNA). Two tandem stop codons were inserted before the trpA terminator to generate the stop reporter construct as previously described (20). In each construct, six histidine codons were introduced before the c1 gene, which enables the capture and enrichment of ribosomes translating the c1 message using affinity chromatography on a Ni column (20). Wild type and smpB mutant strains harboring the constructs were grown in YT medium with 0.2% glucose (to inhibit leakage expression of c1) to an A550 of ~0.3 and were collected by centrifugation. The cell pellet was washed once in YT medium and then grown in the same medium containing 0.02% arabinose for the indicated times to induce expression of c1 mRNA (27). Measurement of RNase R Half-life—Cells were grown in YT medium to an A550 of ~0.3. A portion of the culture was collected for the zero time point and chloramphenicol was added to the remaining culture at 200 μg/ml. Cells were collected at the indicated times, lysed by sonication (13), and assayed by immunoblotting to determine the amount of RNase R remaining. Western Blot Analysis—Proteins were resolved on either 8% (for detection of RNase R) or 12% (for detection of SmpB) SDS-PAGE and subjected to immunoblotting. RNase R and recombinant FLAG-SmpB were detected by purified RNase R antibody (1:10,000 dilution) and anti-FLAG M2 mAbs (1:1000 dilution), respectively. Underexposed films were used for quantitation by Quantity One (Bio-Rad). Northern Blot and RT-PCR Analyses—RNA by gest on O cber 0, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of RNase R by ribosomes 4 was extracted from cells with the RNeasy mini kit according to the manufacturer’s protocol and separated by electrophoresis on 6% or 12% polyacrylamide/7.5 M urea gels. Prehybridization and hybridization was performed as described previously (28). Oligos C3 and T4 (Table 1) were used to probe c1 mRNA and tmRNA, respectively. For RT-PCR, 2 μg of total RNA was used for first-strand cDNA synthesis by M-MLV in the presence of primer R3 (for rnr) or S1 (for smpB). Partial sequences of rnr and smpB were amplified with primers R3 and R4, or S1 and S2 (Table 1), respectively. PCR conditions were as follows: 3 min at 94°C, followed by 30 cycles of 30 s at 94°C; 30 s at 60°C; and 1 min at 72°C. PCR products were resolved on 1.5% agarose gels and visualized by ethidium bromide staining (29). In vitro Proteolysis Assay—HslUV-mediated in vitro degradation of RNase R was performed as described (17). Briefly, 0.1 nM RNase R was mixed with 0.01 nM HslU6 and 0.05 nM HslV12 in proteolysis buffer ( 25 mM HEPES-KOH, pH 7.6, 5 mM KCl, 20 mM MgCl2, 0.032% NP-40, 10% glycerol, 4 mM ATP, 50 mM creatine phosphate and 80 μg/ml creatine kinase), in the presence of 0.1 nM tmRNA and SmpB. The mixtures (100 μl) were incubated at 37°C, and samples were taken at various time points for quantitation of RNase R remaining. To determine the effect of ribosomes on RNase R degradation in vitro, 0.1 nM RNase R was incubated with 0.1 nM Ala-tmRNA, 0.1 nM SmpB, 0.5 nM EF-Tu and 0.2 nM GTP in proteolysis buffer in the presence of 0.5 nM “stop” or “non-stop” ribosomes at 37°C for 10 min. HslU6 (0.01 nM) and 0.05 nM HslV12 were then added and the mixtures were incubated at 37°C for the indicated times. RNase R remaining was detected as described above. Purification of AlaRS and EF-Tu—The alaS and tufB genes were PCR amplified with primers A1, A2 and T5, T6 (Table 1), respectively. The PCR products were purified and digested with NheI and XhoI, or NdeI and BamHI, and then cloned into the corresponding sites on pET28a and pET15b, respectively. Purification of His tagged AlaRS and EF-Tu was carried out according to the manufacturer’s protocol. Aminoacylation of tmRNA in vitro—tmRNA (50 pM) was incubated with 2.5 mM AlaRS in 100 mM Tris-HCl, pH 8.0, 10 mM KCl, 5 mM MgCl2, 2 mM ATP, 10 mM dithioerythritol and 30 mM L-[C] alanine for 1h at 37 °C (30). The RNAs were then extracted with the RNeasy mini kit. Stalled Ribosome Enrichment—smpB mutant strain cells containing the stop or non-stop c1 construct were grown in YT medium with 0.2% glucose to an A550 of ~0.3. The cells were then washed once in YT medium and grown in the same medium containing 0.02% arabinose for 20 min to induce expression of c1 mRNA. Isolation and enrichment of ribosomes containing the translational products of the stop or non-stop c1 gene via Ni column chromatography were performed as previously described (20). Growth Competition—Equal amounts of wild type (Kan) and K544R, ΔC or D278N mutant strain cells, based on absorbance, were mixed and diluted into YT medium. The culture was incubated at 37°C with constant shaking at 200 rpm. Growth was monitored by A550 measurements. After 3 h, the culture still in exponential phase growth, was diluted 1:1000 into fresh YT medium. This represents one cycle (3h) of exponential growth competition. Such cycles were repeated 3 times. Before each cycle, 100 μl of culture was taken, diluted and plated onto YT plates with or without kanamycin. The CFU for each strain was determined at the beginning and end of each cycle. Calculation of by gest on O cber 0, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of RNase R by ribosomes 5 the strains doubling time was carried out as previously described (14). Analysis of Acid-soluble Material Derived from Cellular RNAs—A single colony of wild type, K544R or the ΔC mutant strain was inoculated into 2 ml of M9/0.2% glucose/0.5% Casamino acids medium. After overnight growth, 100 μl of each culture was inoculated into 100 ml of M9/0.2% glucose/0.5% Casamino acids supplemented with 1 mCi/ml of [H]-uridine and 0.1 mM uridine (31). Cultures were grown at 37°C to an A550 of ~0.2 and were collected by centrifugation. The cell pellet was washed once in M9 salts and inoculated into 100 ml of M9/0.2% glucose/0.5% Casamino acids with 0.1 mM uridine to prevent reincorporation of radioactive material. A550 readings were taken to monitor growth. At an A550 of ~0.4, 500 μl portions were removed from the culture and treated with 250 μl of 4 M formic acid (32). After 15 min on ice, samples were centrifuged at maximum speed for 15 min in a Fisher bench top microcentrifuge at 4°C. Half of the supernatant fraction was removed and neutralized with 1 M Tris. Ten milliliters of scintillation fluid was added, and samples were counted in a scintillation counter to determine acid-soluble radioactivity (33). The remaining cells were harvested for total RNA isolation. Measurement of c1 Non-stop mRNA Decay— Wild type cells harboring the c1 non-stop reporter plasmid were grown in YT medium in the absence of glucose to allow low level leakage expression of c1 mRNA. Cells were grown to an A550 of ~0.3 or overnight, and rifampicin (0.45 mg/ml) was then added to inhibit further transcription (20). Samples were taken at the indicated time points and total RNA was extracted. The reporter mRNA was probed with P-labelled oligo C3 (Table 1). Co-immunoprecipitation Assay — Cells were ruptured by sonication in binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.5% NP-40, 1 mM PMSF) containing protease inhibitor cocktail. Anti-FLAG M2-agarose suspension (100 μl per 200 ml of bacterial culture) was then added and incubated overnight with the crude extract at 4°C. The beads were washed 3 times with 500 μl of binding buffer containing 0.5 M NaCl to remove non-specific contaminants, and the bound proteins were eluted with FLAG peptide (1 mg/ml in binding buffer, 50 μl per 200 ml bacterial culture). FLAG-SmpB and RNase R in the eluant and the supernatant fraction were detected by Western blot