Ribosome stalling during translation elongation induces cleavage of mRNA being translated in Escherichia coli

Recently, it has been found that ribosome pausing at stop codons caused by certain nascent peptides induces cleavage of mRNA in Escherichia coli cells (1, 2). The question we addressed in the present study is whether mRNA cleavage occurs when translation elongation is prevented. We focused on a specific peptide sequence (AS17), derived from SecM, that is known to cause elongation arrest. When the crp-crr fusion gene encoding CRP-AS17-IIA(Glc) was expressed, cAMP receptor protein (CRP) proteins truncated around the arrest sequence were efficiently produced, and they were tagged by the transfer-messenger RNA (tmRNA) system. Northern blot analysis revealed that both truncated upstream crp and downstream crr mRNAs were generated along with reduced amounts of the full-length crp-crr mRNA. The truncated crp mRNA dramatically decreased in the presence of tmRNA due to rapid degradation. The 3' ends of truncated crp mRNA correspond well to the C termini of the truncated CRP proteins. We conclude that ribosome stalling by the arrest sequence induces mRNA cleavage near the arrest point, resulting in nonstop mRNAs that are recognized by tmRNA. We propose that the mRNA cleavage induced by ribosome stalling acts in concert with the tmRNA system as a way to ensure quality control of protein synthesis and possibly to regulate the expression of certain genes.


INTRODUCTION
A special bacterial RNA called tmRNA or SsrA RNA is a central player in a unique quality control system during protein synthesis (3)(4)(5). When a ribosome translates to the 3' end of a broken or incomplete mRNA lacking a stop codon (nonstop mRNA), tmRNA charged with alanine enters the Asite of the ribosome to act first as an alanyl-tRNA and then is itself translated. This co-translation reaction (trans-translation) terminates at the stop codon that follows the tmRNA reading frame, releasing both the ribosome and the tagged polypeptide. The tagged polypeptide is recognized and degraded by several ATP dependent proteases. The rescue of the stalled ribosome and the degradation of aberrant polypeptides that are useless and/or potentially harmful to the cell are two well-established biological roles of the tmRNA quality control system (4,5). An additional important role of the tmRNA system is to facilitate the degradation of truncated mRNAs by removing stalled ribosomes and thus allowing 3'-to-5' exonucleases to access the free mRNA 3' end (6). Thus, the tmRNA quality control system not only degrades aberrant polypeptides once produced but also prevents production of aberrant polypeptides through a rapid elimination of damaged mRNAs.
Nonstop mRNAs could be generated either by nuclease cleavages of an mRNA or by incomplete transcription. For example, they are produced as degradation intermediates from mRNAs by 3'-to-5' exonucleolytic digestion (6). The tmRNA-mediated tagging of LacI typically represents the situation where truncated mRNAs are produced by an incomplete transcription due to transcriptional road block (7). A ribosome would reach also the 3' end of an mRNA when a normal stop codon is erroneously translated either in the presence of nonsense suppressor tRNAs (8) or in the presence of misreading drugs (9). Interestingly, the tmRNA system also acts at stop codons to add the degradation tag to full-length proteins when translation termination is prevented by certain nascent polypeptides (10)(11)(12)(13).
During the study on the mechanism of nascent peptide dependent tagging of the full-length protein at stop codons, we have found that ribosome stalling caused by certain peptides leads to mRNA cleavage around the stop codon resulting in nonstop mRNAs and therefore the mRNA cleavage is the cause of trans-translation at stop codons (1). Cleavage of mRNA in response to ribosome stalling at stop codons was also found in ybeL mRNA by Hayes and Sauer (2).
The findings mentioned above have raised a question of whether the cleavage of mRNA occurs at sense codons when translation elongation is prevented. To answer this particular question, we focused on a specific peptide sequence, derived from SecM, that is known to cause stalling of the ribosome during translation elongation (14). In the present study, we constructed the crp-crr fusion genes in which the coding regions of two proteins (CRP and IIA Glc ) are connected by a short nucleotide sequence corresponding to the SecM arrest sequence (14). By using this fusion system, we found that the SecM arrest sequence strongly induces mRNA cleavage resulting in nonstop mRNAs that are recognized by tmRNA. We also found that the last 5 amino acid segment of the arrest sequence is sufficient enough to induce the mRNA cleavage. Neither the bacterial toxin RelE nor the known major endoribonucleases are involved in this cleavage, suggesting that either other endoribonuclease(s) or ribosome itself would be responsible for the mRNA cleavage in response to the ribosome stalling caused by the particular nascent peptides. The mRNA cleavage induced by heated for 5 min at 100°C. For Western blotting, the total extracts of indicated amount were subjected to a 0.1% SDS -12% or 15% PAGE and transferred to Immobilon membrane (Millipore). The membrane was probed with anti-CRP antibodies using ECL system (Amersham Life Science).

Mass spectrometry
The untagged and tagged CRP-GIRAGP proteins were immuno-purified from TA501 (W3110 Dcrp DssrA) and TA481(W3110 Dcrp ssrA DD ) cells carrying pJK208. The cells were grown in 20 ml of LB medium containing 100 mg/ml of ampicillin to OD 600 = 0.6. The cell cultures were centrifuged and washed with 0.1 M NaCl, 10 mM Tris-HCl(pH8.0), 1 mM EDTA. The cell pellets were suspended in 600 ml of buffer A (20 mM Tris-HCl pH 8.0, 0.1 M KCl, 5 mM MgCl 2 , 0.1% Tween-20, 10 mM 2-mercaptoethanol, 10% glycerol, 0.2 mM phenylmethanesulfonyl fuluoride). The cell suspensions were sonicated and centrifuged at 12,000 rpm for 5 min. 400 ml of the supernatants were mixed with 10 ml of anti-CRP antibodies conjugated to agarose beads. Immunoprecipitation was carried out for 2 hrs at 4˚C with gentle agitation. After extensive washing with buffer A, bound proteins were eluted with 10 ml of 100 mM glycine-HCl (pH2.5). The polyclonal anti-CRP antibodies were crosslinked to Protein A-agarose (Roche) with dimethylpimelidate as described (17). For mass spectrometry (MS) analysis, the purified untagged and DD-tagged proteins were separated by 15% SDS-PAGE. The bands were cut out from the gel and a small piece of each band was treated with 0.1 mg of lysyl endopeptidase (Roche) in 20 ml of 25 mM Tris-HCl, pH 9.0 for 12 hr at 37˚C. The digeted peptides were eluted with 300 ml of 50 % acetonitrile, 5 % formic acid and concentrated to 20 ml.  Then, the sample was desalted with zip-tip reverse-phase column, mixed with 1 % a-CHCA (a-cyano-4-hydroxycinnamic acid) in 70 % acetonitrile, and subjected to MALDI/TOF-MS.

RNA analyses
Total RNAs were isolated from cells grown to mid-log phase as described (18). The total RNAs were resolved by 1.5 % agarose-gel electrophoresis in the presence of formaldehyde and blotted onto Hybond-N + membrane (Amersham). The mRNAs were visualized using digoxigenin (DIG) reagents and kits for non-radioactive nucleic acid labeling and detection system (Roche) according to the procedure specified by the manufacturer.
The DIG-labeled DNA probes used were 576 bp probe A corresponding to the crp coding region and 507 bp probe B corresponding to the crr coding region. The DIG-labeled RNA marker III (Roche) was used to estimate the size of RNA bands. The 3' end of crp mRNA and the 5' end of crr mRNA were determined by S1 nuclease assay as described (18). A DNA fragment corresponding to the junction region between the crp and crr was prepared by PCR from pJK208 encoding CRP-GIRAGP-IIA Glc and digested with Sau3AI and HpaII. The 3' end of the resulting 102 bp fragment was labeled with [a-32 P] dGTP by Klenow enzyme and the strands were separated by electrophoresis on a 10 % polyacrylamide gel. The lower strand 32 Plabeled at its Sau3AI 3' end was used as a DNA probe (probe C) to determine the 3' end of crp mRNA. Similarly, a DNA fragment (315 bp) corresponding to the junction region between the crp and crr was prepared by PCR from pJK208 encoding CRP-GIRAGP-IIA Glc and the strands were separated by electrophoresis on a 10 % polyacrylamide gel. The lower strand 32 P-labeled at its 5' end was used as a DNA probe (probe D) to determine the 5' end of crr mRNA. The DNA probe and total RNAs were hybridized, and treated with increasing amounts of S1 nuclease for 15 min at 37°C. The resulting products were analyzed on a 7 % polyacrylamide-8 M urea gel. The ends of mRNAs were identified by using the Maxam-Gilbert A+G and C+T ladders of the DNA probe as references.  (14). A striking feature of this specific segment is that it can inhibit translation elongation even when present within unrelated sequences. This provides us an opportunity to investigate a link between ribosome stalling during translation elongation and tmRNA tagging of the nascent peptide. We first constructed a plasmid (pJK217) carrying a crp-crr fusion gene encoding CRP-AS17-IIA Glc protein in which CRP (cAMP receptor protein) and IIA Glc ORFs are connected by a nucleotide sequence corresponding to AS17 (Fig. 1). Another plasmid (pJK216) carrying a fusion gene encoding CRP-AS17 was also constructed, in which a stop codon was placed just after the AS17 sequence. We also used plasmids pST602 and pJK107 that express CRP-GP and CRP-GP-IIA Glc , respectively.

The
An advantage of the crp-crr fusion system is that both tmRNA tagging and mRNA cleavage can be easily monitored by Western and Northern analyses, respectively. These plasmids were introduced into three isogenic strains regarding the ssrA allele and the expression of proteins was analyzed by by guest on March 24, 2020 http://www.jbc.org/

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Western blotting using anti-CRP antibodies. It is established that a Cterminal GP sequence causes an efficient tmRNA tagging at a stop codon (12,13). Accordingly, CRP-AS17 protein is expected to be efficiently tagged by the tmRNA system because this CRP variant possesses a terminal GP sequence. In fact, CRP-AS17 was highly expressed in the absence of tmRNA while it is markedly reduced in the presence of the wild-type tmRNA due to an efficient tagging and proteolysis ( In the presence of the wild-type tmRNA, the truncated protein was no longer observed while a significant amount of DD-tagged protein was detected in the presence of tmRNA-DD (Fig. 2, lanes 11 and 12). These results imply that the AS17 sequence causes ribosome stalling not only at a stop codon but also at a sense codon by inhibiting translation elongation.

The pentapeptide (IRAGP) is sufficient to cause tmRNA tagging
Among the 17 amino acid SecM arrest sequence, the last hexapeptide GIRAGP seems to be particularly important to inhibit translation elongaton (14). To examine whether this hexapeptide alone could cause translation arrest and tmRNA tagging at a sense codon, we constructed a fusion gene

Identification of the C-terminus of truncated CRP and tagging site
To determine the C-terminus of truncated CRP and tagging site, CRP proteins were purified from cells carrying pJK208 by immuno-precipitation using anti-CRP antibodies conjugated to agarose beads. The purified proteins were subjected to SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining (Fig. 3A). The profile of the purified proteins was essentially the same as that of Western blot analysis shown in Fig. 2. Interestingly, both truncated untagged and DD-tagged CRP proteins consisted of two bands. Each of these bands was excised from the gel and digested in-gel with lysyl endopeptidase that specifically cleaves the peptide bond after lysine residues. The eluted peptides were analyzed by MALDI-TOF mass spectrometry. Representative data for the mass spectrum are shown in Fig. 3B. The upper band of untagged proteins gave two specific signals that correspond to those expected for the Cterminal fragments of CRP-GIRAG and CRP-GIRAGP. The last Pro residue was identified as the translation arrest point during translation of SecM (14). Thus, the truncation of the fusion protein occurs immediately before and after the arrest point. The lower untagged band produced two specific signals corresponding to the expected C-terminal fragments of CRP-G and CRP. This implies that the truncation occurred also before the arrest point. As expected, the DD-tagged bands gave specific signals that correspond to junction peptides containing the C-terminal fragments plus the tag.

The arrest-causing sequence induces mRNA cleavage
We demonstrated previously that the ribosome pausing at stop codons caused by certain nascent peptides generates nonstop mRNAs through  (Fig. 4, lanes 1-6). This suggests that the mRNA cleavage is enhanced by tmRNA.

Determination of the 3'end of the upstream cleavage product
To determine the 3' end of the truncated crp mRNA, an S1 analysis was performed by using a DNA probe that corresponds to the junction region of the crp-crr gene encoding CRP-GIRAGP-IIA Glc . Total RNAs prepared from cells expressing CRP-GIRAGP-IIA Glc were hybridized with the DNA probe C 32 P-labeled at its 3' end. The hybrids were treated with S1 nuclease and the products were analyzed by electrophoresis on a sequencing gel. As shown in Fig. 5, several clusters of S1-resistant bands (referred to as I to V) were detected when RNAs from cells without tmRNA were used (Fig. 5, lane 3). The abundance of these bands except the band V was markedly reduced when RNAs from cells with tmRNA were used (Fig. 5, lane 4).
The cluster III is mapped around the Pro codon of GIRAGP sequence that  (Fig. 6, lanes 1-6). This again suggests that the mRNA cleavage occurs more efficiently in the presence of tmRNA.

Determination of the 5'end of the downstream cleavage product
An S1 analysis was performed to determine the 5' end of the truncated crr mRNA. Total RNAs prepared from cells expressing CRP-GIRAGP-IIA Glc were hybridized with the probe D 32 P-labeled at its 5' end. The hybrids were treated with S1 nuclease and the products were analyzed by electrophoresis on a sequencing gel. In contrast to the 3' end mapping, only one major S1-resistant band was detected and the abundance of this band increased in the presence of tmRNA as expected (Fig. 7, lanes 3 and 4). analyzed by Northern blotting. The disruption of the relEB did not affect the generation of truncated crp and crr mRNAs (Fig. 8, lanes 1-3), suggesting that RelE is not responsible for the mRNA cleavage in response to ribosome stalling. We also tested the effects of mutations in genes encoding RNase E, RNase G, and RNase III on the cleavage reaction. None of these mutations affected the generation of truncated crr mRNA (Fig. 8, lanes 4-6), indicating that these major endonucleases are also not responsible for the cleavage reaction.

DISCUSSION
The major finding of the present study is that inhibition of translation  (21) and the cleavage of ermC mRNA coding for a ribosomal RNA methyltransferase in Bacillus subtilis (22). Although how the present finding is related to these previous observations remains to be studied, cleavage of mRNA appears to be a general event associated with ribosome pausing during translation under various situations.
We demonstrated that the mRNA cleavage in response to a stalled ribosome does not require tmRNA itself because it occurs in the absence of tmRNA. However, we also found that the mRNA cleavage is enhanced in the presence of tmRNA. This suggests that tmRNA interacts with the stalled ribosome prior to the cleavage and affect the cleavage reaction. In fact, it is known that a significant fraction of tmRNA is associated with 70S ribosome in cells (23,24). In particular, it should be noted that tmRNA interacts strongly with 16S rRNA and several stretches in tmRNA are complementary to the sequences in 16S rRNA (23).Thus, it is quite possible that tmRNA modulates the conformation of the stalled ribosome through the interaction with 16S RNA to enhance the cleavage reaction.
Further study is necessary to understand how tmRNA stimulates the mRNA cleavage reaction within the stalled ribosome. There are a number of systems in which specific regulatory nascent peptides may affect translation elongation and/or termination presumably by interacting with ribosome. In most of these cases, the peptides appear to Recently, it was shown that several bacterial toxins induce or exhibit endonucleolytic cleavage of mRNAs with the codon or sequence specificity (19,(30)(31)(32). For example, RelE induces endonucleolytic cleavage of mRNAs bound to ribosomes at UAA and other codons in response to a stalled ribosome (19). However, the cleavage of ybeL mRNA at a stop codon was observed in various toxin-deficient strains including DrelE, DyoeB, DyafQ, DmazF, and DchpBK (2). We also observed that the disruption of relEB gene did not affect the mRNA cleavage in response to ribosome stalling at a stop codon (1) and at a sense codon (Fig. 8). In addtion, we showed that the mRNA cleavage in response to ribosome stalling occurs in strains lacking either RNase E, RNase G,or RNase III       were analyzed. Both nucleotide and amino acid sequences around the GIRAGP sequence (bold letter and boxed) are shown on the right. The asterisks are the 5' ends identified by the S1 analysis.   Derivative of pHA7 carrying the crp-crr fusion gene This work encoding CRP-AS17 pJK217 Derivative of pHA7 carrying the crp-crr fusion gene This work encoding CRP-AS17-IIA Glc pJK208 Derivative of pHA7 carrying the crp-crr fusion gene This work encoding CRP-GIRAGP-IIA Glc pJK220 Derivative of pHA7 carrying the crp-crr fusion gene This work encoding CRP-IRAGP-IIA Glc pJK219 Derivative of pHA7 carrying the crp-crr fusion gene This work encoding CRP-RAGP-IIA Glc _________________________________________________________________________