A-Site mRNA Cleavage Is Not Required for tmRNA-Mediated ssrA-Peptide Tagging

In Escherichia coli, prolonged translational arrest allows mRNA degradation into the A site of stalled ribosomes. The enzyme that cleaves the A-site codon is not known, but its activity requires RNase II to degrade mRNA downstream of the ribosome. This A-site mRNA cleavage process is thought to function in translation quality control because stalled ribosomes are recycled from A-site truncated transcripts by the tmRNA-SmpB “ribosome rescue” system. During rescue, the tmRNA-encoded ssrA peptide is added to the nascent chain, thereby targeting the tagged protein for degradation after release from the ribosome. Here, we examine the influence of A-site mRNA cleavage upon tmRNA-SmpB activity. Using a model transcript that undergoes stop-codon cleavage in response to inefficient translation termination, we quantify ssrA-peptide tagging of the encoded protein in cells that contain (rnb+) or lack (Δrnb) RNase II. A-site mRNA cleavage is reduced approximately three-fold in Δrnb backgrounds, but the efficiency of ssrA-tagging is identical to that of rnb + cells. Additionally, pulse-chase analysis demonstrates that paused ribosomes recycle from the test transcripts at similar rates in rnb + and Δrnb cells. Together, these results indicate that A-site truncated transcripts are not required for tmRNA-SmpB-mediated ribosome rescue and suggest that A-site mRNA cleavage process may play a role in other recycling pathways.


Introduction
Non-stop mRNAs pose a significant molecular quality control problem for all organisms [1]. Non-stop transcripts lack in-frame stop codons and therefore encode incomplete polypeptides that could be deleterious for the cell. Furthermore, ribosomes stall at the 39-ends of non-stop mRNA because translation termination and subsequent ribosome recycling require an intact A-site stop codon. All bacteria use transfer-messenger RNA (tmRNA) and SmpB to recycle ribosomes from non-stop messages [1,2]. tmRNA is a bi-functional RNA that acts as both a tRNA and mRNA to ''rescue'' stalled ribosomes and target the associated polypeptides for rapid degradation [3]. The tRNA-like domain of tmRNA is aminoacylated with alanine and allows recognition of stalled ribosomes [3,4]. After the nascent peptide is transferred to tmRNA, the non-stop transcript is released from the ribosome and translation resumes using a short reading frame within tmRNA. In this manner, the tmRNA-encoded ssrA peptide is added to the C-terminus of the nascent chain. The ssrA peptide is recognized by several proteases, which rapidly degrade tagged proteins after release from the ribosome [3,5,6,7]. Because the ssrA coding sequence is terminated with a stop codon, the rescued ribosome is able to undergo normal translation termination and recycling. SmpB is a small tmRNA-binding protein that coordinates the tRNA and mRNA functions of tmRNA [8,9]. The flexible C-terminal tail of SmpB is required for ribosome binding, and recent structural studies indicate that this region mimics the missing A-site codon:anticodon helix on stalled ribosomes [10,11,12]. SmpB is also critical for proper presentation of the tmRNA ''resume'' codon in the A-site after release of the non-stop message [13,14]. Thus, tmRNA-SmpB acts as a translational quality control system that responds to non-processive protein synthesis. Because the tmRNA-SmpB complex provides stalled ribosomes with a stop codon in trans, this process is often termed trans-translation in the literature [15].
tmRNA-SmpB also rescues ribosomes that pause at internal sites within full-length messages. SsrA-peptide tagging activity has been reported for translational pauses that occur at clusters of rare codons [16,17,18], in response to specific nascent peptide sequences [19,20,21,22], and during acute starvation for amino acids [23,24]. In some instances, translational arrest results in cleavage of the A-site codon [24,25,26]. These results suggest that A-site mRNA cleavage is induced to generate non-stop mRNA and promote tmRNA-SmpB recruitment to paused ribosomes. This model is supported by in vitro studies showing that transtranslation occurs more rapidly at ribosomes that have no A-site codon [27]. However, A-site cleaved transcripts are only detected in mutants that lack functional tmRNA-SmpB [25,26]. One explanation for the apparent lack of A-site mRNA cleavage in wild-type cells is that tmRNA-SmpB recycles stalled ribosomes from cleaved transcripts thereby promoting their rapid turnover [25,26,28]. Additionally, not all translational arrests induce A-site mRNA cleavage. In several instances, transcripts are degraded to the 39-leading edge of the paused ribosome, leaving 12 -18 nucleotides downstream of the A-site codon [21,23,26]. Therefore, although truncated mRNA is an important determinant for tmRNA-SmpB activity, it is not clear that transcripts must be truncated in the A-site codon for efficient trans-translation.
The 39-to-59 exoribonuclease RNase II (encoded by the rnb gene) is required for efficient A-site mRNA cleavage in E. coli [29]. Translational arrest in E. coli Drnb mutants produces transcripts that are truncated to a position 12 nucleotides downstream of the A-site codon [29]. This +12 truncation site probably corresponds to the ''toeprint'' of the paused ribosome on mRNA, suggesting that another nuclease(s) degrades transcripts to this position in the absence of RNase II. Notably, RNase II cannot degrade mRNA into the ribosome A site and therefore its role in A-site cleavage must be indirect [29,30]. We have proposed that RNase II degrades mRNA downstream of the paused ribosome, which then facilitates the activity of the actual A-site nuclease. In accord with this model, A-site cleavage is suppressed by stable mRNA structures that are resistant to degradation by RNase II [29,31,32]. In this study, we modulate A-site mRNA cleavage to determine its importance for tmRNA-SmpB mediated ribosome rescue. We find that ssrA-peptide tagging is indistinguishable in rnb + and Drnb genetic backgrounds. Moreover, the rates of peptidyl-tRNA turnover from stalled ribosomes are similar in rnb + and Drnb cells, indicating the ribosome recycling is largely unaffected by the A-site mRNA cleavage process. Together, these results suggest that mRNA degradation to the 39-edge of the stalled ribosome is sufficient for efficient tmRNA-SmpB rescue activity.

Bacterial strains and plasmids
All bacterial strains were derivatives of E. coli strain X90 and are listed in Table 1. Deletions of rnb, pnp and rnr have been described previously [21]. These alleles were introduced into strains CH12, CH113 and CH2385 by phage P1-mediated generalized transduction [33]. The rnb rnr double mutant was constructed by removing the kanamycin-resistance cassette [34] from the Drnb::kan allele to create CH113 Drnb, followed by phage P1mediated transduction of the other gene deletions into the Drnb background. All other gene deletion constructs were transduced from the Keio collection [35] into strains CH113 or CH113 Drnb. All strains were subjected to whole-cell PCR to confirm chromosomal structure. Plasmid pHis 6 -YbeL-PP was constructed by amplification of ybeL-PP using oligonucleotides ybeL-his6-Nco (59 -TAC CAT GGG CAG CAG CCA TCA TCA TCA  TCA TCA TTC TAG TCA TAT GAA CAA GGT TGC TCA) and pET-Eco (59 -CGT CTT CAA GAA TTC TCA TGT TTG ACA GC), followed by digestion with NcoI/EcoRI and ligation to plasmid pET11d.

Protein and RNA analysis
His 6 -tagged proteins were purified by Ni 2+ -affinity chromatography as described [36]. Cultures were grown to mid-log phase (OD 600 ,0.5) and harvested over ice. Cells were collected by centrifugation and frozen at 280uC. Frozen cells were broken by freeze-thaw in urea lysis buffer [50% urea 2 10 mM Tris-HCl (pH 8.0) 2 150 mM NaCl] and lysates clarified by centrifugation at 13,000 rpm for 15 min. Lysates were incubated with Ni 2+ -NTA agarose resin on a rotisserie for 1 hr at room temperature. The resin was washed with 20 mL of lysis buffer supplemented with 20 mM imidazole. His 6 -tagged proteins were eluted with lysis buffer supplemented with 250 mM imidazole. Protein samples were resolved by SDS-PAGE and stained with Coomassie blue or subjected to immunoblot analysis with polyclonal antibodies to the ssrA(DD) peptide. Fluorescent secondary antibodies (anti-rabbit) were obtained from Rockland Immunochemicals. Stained gels and immunoblots were visualized and quantified using the Odyssey infrared imager and software package (LiCor). RNA was isolated and analyzed as described previously [21,37]. Transcripts were analyzed by northern blot hybridization using [ 32 P]-labeled oligonucleotide probe-T7-SD (59 -GTA TAT CTC CTT CTT AAA GTT AAA C) as described [37]. A-site truncation products were quantified using the Quantity One software package (BioRad). The reported values for percent A-site truncated mRNA represent the mean 6 standard error of the mean (SEM) for three to six independent experiments.
Pulse-chase analysis E. coli cells were grown to exponential phase in MOPS-buffered defined media [38], pulse labeled with 20 mCi/mL of [ 35 S]-Lmethionine/L-cysteine (MP Biomedicals 2 1175 Ci/mmol) and chased with 0.2 mg/mL unlabeled L-methionine/L-cysteine as described [37,39]. RNA was isolated and run on acid-urea polyacrylamide gels as described [39]. Gels were dried and visualized by phosphorimaging. Radiolabeled peptidyl-tRNAs were quantified using Quantity One, and double-exponential decay equations were fitted to the data to estimate rates of peptidyl-tRNA turnover. Reported values represent average rates 6 SEM for two independent experiments.

A-site mRNA cleavage is not required for tmRNAmediated peptide tagging
The correlation between A-site mRNA cleavage in ssrA 2 cells and ssrA-peptide tagging activity in ssrA + cells suggests that these processes are linked functionally. To test this model, we asked whether suppression of A-site mRNA cleavage leads to reduced peptide tagging. We chose the previously characterized YbeL-PP protein from E. coli as a model system to study site-specific translational arrest [25,37,40]. YbeL-PP carries a C-terminal Pro-Pro nascent peptide motif that interferes with translation termination [40,41,42]. As a consequence, the ybeL-PP stop codon is cleaved to generate a non-stop message [25], and the nascent chain is tagged with the ssrA peptide [40]. To facilitate the analysis of cleaved ybeL-PP transcripts, we used the flag-(m)ybeL-PP minigene construct, which encodes a FLAG epitope fused to the Cterminal 49 residues of YbeL-PP (Fig. 1A). The Pro-Pro motif induces ribosome arrest in all genetic contexts tested, and A-site cleavage and ssrA-peptide tagging activities are essentially identical for the full-length and mini-gene ybeL-PP constructs [25,29,37,40]. Full-length flag-(m)ybeL-PP transcripts predominate in ssrA + cells, whereas approximately 30% of the message is truncated at the stop codon in ssrAcells ( RNase II is required for efficient A-site mRNA cleavage activity, and flag-(m)ybeL-PP transcripts are truncated +12 nucleotides downstream of the stop codon when expressed in E. coli ssrA 2 cells that lack RNase II (encoded by the rnb gene) (Figs. 1A & 1B) [29]. Quantification of the stop-codon truncated transcripts indicates that these products are approximately three-fold less abundant in ssrA 2 Drnb cells (8.161.9%) compared with ssrA 2 rnb + cells (Fig.  1C).
To determine whether diminished A-site cleavage correlates with decreased tmRNA-SmpB activity, we examined ssrA-peptide tagging of full-length YbeL-PP proteins in E. coli ssrA(DD) cells.

Role of other RNases and RNA helicases in mRNA processing and tmRNA-mediated peptide tagging
The presence of +12 truncated transcripts in Drnb cells suggests that another unidentified RNase degrades mRNA to this position in the absence of RNase II. To identify the enzyme responsible for this activity, we examined mRNA processing in strains that are deleted for known RNase genes. RNase gene deletions were transferred into E. coli ssrA 2 Drnb cells by transduction and the effects on flag-(m)ybeL-PP processing were assessed by northern blot analysis. Deletion of genes encoding RNase I (rna), RNase D (rnd), RNase T (rnt), RNase PH (rph), RNase Z (elaC), RNase LS (rnlA) and RNase G (rng) has only modest effects on A-site mRNA cleavage in the ssrA 2 background and +12 cleavage in the ssrA 2 Drnb background (Fig. 3A). Additionally, deletion of the Cterminus of RNase E (rne515), which organizes the multienzyme RNA ''degradosome'' [47], does not change the pattern of transcript processing (Fig. 3A). However, deletion of rnc (encoding RNase III) in the ssrA 2 Drnb background restores A-site mRNA cleavage (Fig. 3A, lowest panel). This latter effect is likely due to ,10-fold up-regulation of PNPase expression in Drnc mutants [48]. We have previously shown that PNPase overexpression is sufficient to restore A-site mRNA cleavage in Drnb mutants [29], suggesting that the Drnc mutation may have the same effect. Quantification of ssrA(DD)-tagged His 6 -YbeL-PP proteins from the RNase deletion strains showed that tagging activity is not significantly altered in most instances (Fig. 3B). However, ssrA(DD)-tagging efficiency was slightly, but reproducibly, reduced in Drnb Drnt mutants (Fig. 3B).
RNA helicases are important for the regulation of mRNA translation in eukaryotic cells and could play similar roles in bacteria [49]. In fact, the DEAD-box helicase, RhlB, is found within the RNA degradosome, where it facilitates mRNA turnover by unwinding secondary structures [50]. Based on these observations, we screened four DEAD-box helicases, RhlB, RhlE, HrpA and DeaD, to determine whether these enzymes influence the A-   in combination with the individual helicase knockouts did not change the cleavage patterns (Fig. 4A). We also moved the helicase deletions into ssrA(DD) rnb + and ssrA(DD) Drnb backgrounds, but found that these enzymes have little to no effect on ssrA-peptide tagging efficiency (Fig. 4B). Together, these results indicate that RNase II is required for A-site cleavage and that other known RNases and RNA helicases appear to play no role in this process.
YafO toxin does not catalyze +12 cleavage during ribosome arrest The results presented above also indicate that none of the tested exoribonucleases are individually required for +12 processing in ssrA 2 Drnb cells. Inouye and colleagues recently characterized a type II toxin/antitoxin (TA) module from E. coli that encodes an RNase with an activity that is similar to the +12 cleavage activity described here. YafO acts on translation initiation complexes to cleave mRNA near the +15 position with respect to the P-site AUG initiation codon [51]. This site corresponds to +12 processing in our system, suggesting that YafO may be activated in response to ribosome pausing. We deleted yafO in ssrA 2 and ssrA 2 Drnb backgrounds and examined the effect on flag-(m)ybeL-PP transcript processing, but found no changes in transcript profiles between yafO + and DyafO strains (Fig. 5). Thus, the YafO toxin is not required for +12 cleavage during ribosome arrest.

RNase II activity does not accelerate ribosome recycling
Because A-site cleavage has no discernable effect on ssrApeptide tagging, this mRNA processing is probably not required for tmRNA-SmpB-mediated ribosome rescue. However, there are at least two other ribosome rescue systems in E. coli [52,53,54], raising the possibility that A-site cleavage facilitates ribosome recycling through an alternative pathway. Therefore, we measured the rates of paused ribosome recycling in rnb + and Drnb backgrounds. Because ribosomes pause during the termination of ybeL-PP translation, they carry nascent chains that are covalently linked to P-site tRNA 2 Pro (Fig. 1A). We have previously shown that peptidyl prolyl-tRNA 2 Pro accumulates in response to translational arrest and can be exploited as a biochemical marker of paused ribosomes [37,39]. We pulse labeled nascent chains with [ 35 S]labeled methionine/cysteine and monitored their turnover during a chase with excess unlabeled amino acids. As reported previously, peptidyl prolyl-tRNA 2 Pro turns-over more rapidly in ssrA + cells compared to ssrA 2 cells (Table 2) [37], consistent with the role of tmRNA-SmpB in ribosome rescue. Somewhat unexpectedly, peptidyl prolyl-tRNA 2 Pro turnover is slower in wild-type ssrA + rnb + cells compared to the ssrA + Drnb cells (Figs. 6A & 6B, Table 2). However, in the ssrA 2 background, deletion of rnb results in a slightly longer half-life (although almost within error) for peptidyl prolyl-tRNA 2 Pro (Table 2). Together, these data indicate that the  A-site mRNA cleavage process has a modest effect on the rate of paused ribosome recycling.

Discussion
The original model of trans-translation postulated that ribosome arrest at the 39-end of nonstop mRNA is the signal for tmRNA-SmpB recruitment [3]. This conclusion is supported by subsequent in vitro studies by Ehrenberg and colleagues. The latter work shows that nascent chain transfer to tmRNA occurs most rapidly when 0-6 nucleotides are present downstream of the P-site codon, and that transfer rates diminish with longer transcripts [27]. Although the initial in vitro rate of trans-transfer is close to zero when there are 15 nucleotides downstream of the P-site codon (equivalent to +12 processing in our system), these same reactions approach 50% completion after 1 s of incubation [27]. Our results suggest that degradation of mRNA to the 39-edge of stalled ribosomes is sufficient for tmRNA-SmpB-mediated rescue in vivo. Perhaps tmRNA-SmpB induces a slow conformational change in the ribosome that allows trans-translation to occur upon prolonged arrest. Structural studies indicate that the C-terminal tail of SmpB interacts with 30S A site, where it is thought to mimic the missing codon:anticodon mini-helix [9,12,55]. Therefore, A-site mRNA must presumably be displaced to accommodate tmRNA-SmpB binding. Although there appears to be a discrepancy between the in vitro and in vivo requirements for trans-translation, it is possible that an unknown cellular factor enhances trans-translation when transcripts extend beyond the stalled ribosome A site. We note that there are several examples of ribosome arrest that do not induce A-site mRNA cleavage [18,19,20,23], suggesting that longer truncated transcripts represent a major pathway for ribosome rescue. A rigorous test of this model awaits identification of the nuclease(s) responsible for +12 mRNA processing.
Most E. coli messages are thought to be first recognized and cleaved by RNase E to produce fragments that are subsequently degraded by 39-to-59 exoribonucleases [43]. RNase E preferentially binds monophosphate groups at the 59-ends of transcripts, and therefore the bulk flow of mRNA degradation proceeds with 59-to-39 polarity even though E. coli lacks known 59-to-39 exoribonucleases. This strategy minimizes the production of translatable non-stop messages during mRNA turnover. Moreover, mRNA turnover is typically processive without the accumulation of decay intermediates. However, the results presented here and elsewhere show that paused ribosomes stabilize partially degraded transcripts that lack 39-ends [18,19,20,21,23,24,25,26,28,56]. It is unclear whether these fragments represent normal decay intermediates that are stabilized by stalled ribosomes, or whether they accumulate because the 59to-39 degradation pathway is disrupted by queued ribosomes. In either case, paused ribosomes interfere with processive mRNA decay. When we first discovered A-site mRNA cleavage, we proposed that this activity was critical for mRNA turnover because  it would accelerate ribosome recycling and expose truncated transcripts to exoribonucleases [25]. Although that original conclusion is not supported by the present study, tmRNA-SmpB activity does indeed hasten mRNA decay. Karzai and colleagues have shown that ribosome rescue leads to rapid degradation of truncated transcripts by RNase R [57,58]. The results presented here now suggest that +12 cleavage is sufficient for these tmRNA-SmpB-dependent effects on mRNA turnover. If +12 truncated transcripts are sufficient for the tmRNA-SmpB activity, then what is the functional significance of A-site mRNA cleavage? Because A-site cleavage is only detected in ssrA (or smpB) mutants, it may be a response to prolonged translational arrest in the absence of ribosome rescue. This model suggests that A-site cleavage could play a role in alternative ribosome rescue mediated by ArfA [53]. ArfA functions as a back-up ribosome rescue system that is only deployed when tmRNA-SmpB is overwhelmed or incapacitated [59,60]. ArfA binds to stalled ribosomes and induces nascent chain release by recruiting release factor-2 (RF-2) [61,62]. Because RF-2 activity requires an intact stop codon, it is possible that ArfA binds the 30S A site to mimic a UGA stop codon. Consistent with this model, we have found that ArfA-mediated rescue activity requires an incomplete A-site codon in vivo (F.G.S. and C.S.H. unpublished results). However, if A-site mRNA cleavage is required for the ArfA pathway, then E. coli ssrA 2 Drnb cells should be inviable just like E. coli ssrA 2 DarfA mutants [53,63]. Another possibility is that yet another rescue pathway mediated by YaeJ (ArfB) is upregulated to compensate in these genetic backgrounds [52,54]. Although YaeJ/ArfB-mediated rescue appears to be quantitatively less important in E. coli, it could represent a major rescue pathway in bacteria that lack ArfA. Additionally, because the ribosome arrest studied here is due to consecutive prolyl residues in the nascent chain, it is possible that elongation factor-P could play a role in ribosome-restart to allow RF-mediated termination [64,65,66]. Clearly, further study will be required to ascertain the relative importance and functional interactions between these recycling pathways.