Nonstop mRNA Research Articles

Trans-translation: Findings and hypotheses

Trans-translation is a unique process that switches the synthesis of a polypeptide hain encoded by a nonstop mRNA to the mRNA-like domain of tmRNA. The process is used in bacterial cells for rescuing the ribosomes arrested during translation of nonstop mRNA and directing this mRNA and the polypeptide product for degradation. Activity of tmRNA is essential for bacterial survival under adverse conditions, the quality control of translation, and the regulation of certain physiological pathways. The review focuses on recent advances in trans-translation studies. Details of the tmRNA-SmpB interaction and the structures of early ribosomal complexes are characterized, the causes of the appearance of an empty A site in the translating ribosome and possible mechanisms of the stalled ribosome recognition and resume codon determination are discussed, and the proteins degrading nonstop mRNAs and tagged peptides are considered.

Co‐evolution of multipartite interactions between an extended tmRNA tag and a robust Lon protease in Mycoplasma

Messenger RNAs that lack in-frame stop codons promote ribosome stalling and accumulation of aberrant and potentially harmful polypeptides. The SmpB-tmRNA quality control system has evolved to solve problems associated with non-stop mRNAs, by rescuing stalled ribosomes and directing the addition of a peptide tag to the C-termini of the associated proteins, marking them for proteolysis. In Escherichia coli, the ClpXP system is the major contributor to disposal of tmRNA-tagged proteins. We have shown that the AAA+ Lon protease can also degrade tmRNA-tagged proteins, but with much lower efficiency. Here, we present a unique case of enhanced recognition and degradation of an extended Mycoplasma pneumoniae (MP) tmRNA tag by the MP-Lon protease. We demonstrate that MP-Lon can efficiently and selectively degrade MP-tmRNA-tagged proteins. Most significantly, our studies reveal that the larger (27 amino acids long) MP-tmRNA tag contains multiple discrete signalling motifs for efficient recognition and rapid degradation by Lon. We propose that higher-affinity multipartite interactions between MP-Lon and the extended MP-tmRNA tag have co-evolved from pre-existing weaker interactions, as exhibited by Lon in E. coli, to better fulfil the function of MP-Lon as the sole soluble cytoplasmic protease responsible for the degradation of tmRNA-tagged proteins.

Kinetics of Paused Ribosome Recycling in Escherichia coli

The bacterial tmRNA·SmpB system recycles stalled translation complexes in a process termed ‘ribosome rescue.’ tmRNA·SmpB specifically recognizes ribosomes that are paused at or near the 3′ end of truncated mRNA; therefore, nucleolytic mRNA processing is required before paused ribosomes can be rescued from full-length transcripts. Here, we examine the recycling of ribosomes paused on both full-length and truncated mRNAs. Peptidyl-tRNAs corresponding to each paused translation complex were identified, and their turnover kinetics was used to estimate the half-lives of paused ribosomes in vivo. Ribosomes were paused at stop codons on full-length mRNA using a nascent peptide motif that interferes with translation termination and elicits tmRNA·SmpB activity. Peptidyl-tRNA turnover from these termination-paused ribosomes was slightly more rapid in tmRNA+ cells (T1/2=22±2.2 s) than in ΔtmRNA cells (T1/2=32±1.6 s). Overexpression of release factor (RF) 1 greatly accelerated peptidyl-tRNA turnover from termination-paused ribosomes in both tmRNA+ and ΔtmRNA cells, whereas other termination factors had little or no effect on recycling. In contrast to inefficient translation termination, ribosome recycling from truncated transcripts lacking in-frame stop codons was dramatically accelerated by tmRNA·SmpB. However, peptidyl-tRNA still turned over from nonstop-paused ribosomes at a significant rate (t1/2=61±7.3 s) in ΔtmRNA cells. Overexpression of RF-1, RF-3, and ribosome recycling factor in ΔtmRNA cells failed to accelerate ribosome recycling from nonstop mRNA. These results indicate that tmRNA·SmpB activity is rate limited by mRNA cleavage, and that RF-3 and ribosome recycling factor do not constitute a tmRNA-independent rescue pathway, as previously suggested. Peptidyl-tRNA turnover from nonstop-paused ribosomes in ΔtmRNA cells suggests the existence of another uncharacterized ribosome rescue pathway.

Analysis of nonstop mRNA translation in the absence of tmRNA inEscherichia coli

tmRNA, a product of ssrA gene, plays a crucial role in the quality control system that eliminates aberrant products of nonstop mRNAs in prokaryotes. Although tmRNA recycles ribosomes stalled at the 3' end of nonstop mRNAs, the fate of ribosomes that stall at the 3' end in the absence of tmRNA has not been extensively examined. Here we report our analysis of the translation status of nonstop mRNAs. Polysome analysis showed that nonstop mRNAs were translated efficiently, and peptidyl-tRNA was not found in any fraction in a DeltassrA strain. In vitro translation experiments using PURESYSTEM revealed that ribosomes translating nonstop mRNAs were dissociated from the 3' end of mRNA, and the peptidyl-tRNA was only weakly hydrolyzed in the monosome. These results suggest that the peptidyl-tRNA of a nonstop mRNA is hydrolyzed by an unknown factor(s) in vivo, thereby allowing a nonstop mRNA to be translated as efficiently as a normal mRNA. Possible factors involved in the hydrolysis of the peptidyl-tRNAs of nonstop mRNAs are discussed.

Nascent Peptide-dependent Translation Arrest Leads to Not4p-mediated Protein Degradation by the Proteasome

The potentially deleterious effects of aberrant mRNA lacking a termination codon (nonstop mRNA) are ameliorated by translation arrest, proteasome-mediated protein destabilization, and rapid mRNA degradation. Because polylysine synthesis via translation of the poly(A) mRNA tail leads to translation arrest and protein degradation by the proteasome, we examined the effects of other amino acid sequences. Insertion of 12 consecutive basic amino acids between GFP and HIS3 reporter genes, but not a stem-loop structure, resulted in degradation of the truncated green fluorescent protein (GFP) products by the proteasome. Translation arrest products derived from GFP-R12-FLAG-HIS3 or GFP-K12-FLAG-HIS3 mRNA were detected in a not4Delta mutant, and MG132 treatment did not affect the levels of the truncated arrest products. Deletion of other components of the Ccr4-Not complex did not increase the levels of the translation arrest products or reporter mRNAs. A L35A substitution in the Not4p RING finger domain, which disrupted its interaction with the Ubc4/Ubc5 E2 enzyme and its activity as an ubiquitin-protein ligase, also abrogated the degradation of arrest products. These results suggest that Not4p, a component of the Ccr4-Not complex, may act as an E3 ubiquitin-protein ligase for translation arrest products. The results let us propose that the interaction between basic amino acid residues and the negatively charged exit tunnel of the ribosome leads to translation arrest followed by Not4p-mediated ubiquitination and protein degradation by the proteasome.

Codon Optimization Increases Steady-State mRNA Levels in Aspergillus oryzae Heterologous Gene Expression

We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3' rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in A. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.

One SmpB molecule accompanies tmRNA during its passage through the ribosomes

tmRNA and SmpB are the main participants of trans-translation, a process which rescues the ribosome blocked during translation of non-stop mRNA. While a one-to-one stoichiometry of tmRNA to the ribosome is generally accepted, the number of SmpB molecules in the complex is still under question. We have isolated tmRNA–ribosome complexes blocked at different steps of the tmRNA path through the ribosome and analyzed the stoichiometry of the complexes. Ribosome, tmRNA and SmpB were found in equimolar amount in the tmRNA–ribosome complexes stopped at the position of the 2nd, 4th, 5th or the 11th codons of the coding part of the tmRNA.

Cleavage of mRNAs and role of tmRNA system under amino acid starvation in Escherichia coli

We have shown previously that ribosome stalling during translation caused by various reasons leads to mRNA cleavage, resulting in non-stop mRNAs that are eliminated in a tmRNA-dependent manner. Amino acid starvation is a physiological condition in which ribosome stalling is expected to occur more frequently. Here we demonstrate that mRNA cleavage is induced by amino acid starvation, resulting in accumulation of truncated mRNAs in cells lacking tmRNA. The truncated mRNAs are eliminated in wild-type cells, indicating that the tmRNA system rapidly degrade the truncated mRNAs. The cleavage pattern of model mRNAs in which serine codons were replaced with threonine codons indicated that mRNA cleavage occurs near serine codons in response to serine starvation. Cells lacking all of the five known toxin loci were proficient in mRNA cleavage, showing that toxin-antitoxin systems are not responsible for the cleavage. A mild serine starvation caused a significant growth inhibition in cells lacking tmRNA but not in wild-type cells. The ribosome-mediated mRNA cleavage along with the tmRNA system is an important mechanism that enables cells to adapt to amino acid starvation conditions.

Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components

BackgroundMembers of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis.ResultsExtensive BLAST searches confirm that Hbs1p and eRF3 are limited to eukaryotes, while Dom34p and eRF1 (a/eRF1) are universal in eukaryotes and archaea. Ski7p appears to be restricted to a subset of Saccharomyces species. Alignments show that Dom34p does not possess the characteristic class-1 RF minidomains GGQ, NIKS and YXCXXXF, in line with recent crystallographic analysis of Dom34p. Phylogenetic trees of the protein families allow us to reconstruct the evolution of mRNA surveillance mechanisms mediated by these proteins in eukaryotes and archaea.ConclusionWe propose that the last common ancestor of eukaryotes and archaea possessed Dom34p-mediated no-go decay (NGD). This ancestral Dom34p may or may not have required a trGTPase, mostly like a/eEF1A, for its delivery to the ribosome. At an early stage in eukaryotic evolution, eEF1A was duplicated, giving rise to eRF3, which was recruited for translation termination, interacting with eRF1. eRF3 evolved nonsense-mediated decay (NMD) activity either before or after it was again duplicated, giving rise to Hbs1p, which we propose was recruited to assist eDom34p in eukaryotic NGD. Finally, a third duplication within ascomycete yeast gave rise to Ski7p, which may have become specialised for a subset of existing Hbs1p functions in non-stop decay (NSD). We suggest Ski7p-mediated NSD may be a specialised mechanism for counteracting the effects of increased stop codon read-through caused by prion-domain [PSI+] mediated eRF3 precipitation.

Functional SmpB-Ribosome Interactions Require tmRNA

Small protein B (SmpB) is a requisite component of the transfer messenger RNA (tmRNA)-mediated bacterial translational quality control system known as trans-translation. The initial binding of tmRNA and its subsequent accommodation into the ribosomal A-site are activities intimately linked to SmpB protein function. From a mechanistic perspective, two key unanswered questions that require further investigation are: 1) what constitutes a stalled ribosome recognition complex and 2) does SmpB pre-bind ribosomes to recruit tmRNA. We have assessed, both in vivo and in vitro, the nature and stability of free SmpB interactions with stalled ribosomes and examined whether these interactions are functionally relevant. We present evidence to demonstrate that interaction of free SmpB with ribosomes is salt sensitive and significantly more labile than interaction of the SmpB.tmRNA complex with ribosomes. Upon dissociation of 70 S ribosomes SmpB partitions primarily with tmRNA rather than ribosomal subunits. This finding is consistent with biochemical and structural data demonstrating that tmRNA is the high-affinity binding partner of SmpB. Moreover, we show that under normal physiological conditions roughly similar numbers of SmpB and tmRNA molecules are present in cells. Our investigations also reveal that upon induction of a nonstop mRNA, SmpB is enriched in stalled ribosome fractions only in the presence of tmRNA. Based on these findings, we conclude that SmpB does not pre-bind stalled ribosome and that functional SmpB-stalled ribosome interactions require tmRNA. We propose that a 1:1:1 complex of SmpB.tmRNA.EF-Tu(GTP) recognizes and binds a stalled ribosome to initiate trans-translation.

Molecular characterization of the first intact Transib transposon from Helicoverpa zea

Transib is a superfamily of DNA transposons recently reconstructed in silico from degenerate elements in the genomes of Drosophila melanogaster and Anopheles gambiae. Here we report characterization of the first intact Transib transposon designated Hztransib from Helicoverpa zea. At least two Hztransib copies (one full-length, one internally-deleted) exist in the genomes of a midgut cell line and a laboratory strain of H. zea. The full-length Hztransib has 3518 bp including a 5′ terminal inverted repeat (TIR) of 552 bp, a promoter sequence of 381 bp, an intact open reading frame encoding 507 amino acids, and a 3′ TIR of 502 bp, and is flanked by 5-bp (CGTCG) target site duplications. The full-length Hztransib is transcriptionally active, producing an 3′-truncated mRNA lacking a termination codon (known as nonstop mRNA) due to alternative polyadenylation in two somatic tissues (midgut and fat body) and one germline tissue (ovary). A BLAST search with the deduced Hztransib transposase identified 51 novel Transib elements from 11 insect genomes. While the full-length Hztransib inserts into the 5′-flanking region of a xenobiotic-metabolizing P450 gene CYP6B8 in the midgut cell line, it does not insert into the 5′-flanking region of CYP6B8 in the laboratory strain. Such an insertion dimorphism, together with its complete structural features and mRNA transcripts, demonstrates that the full-length Hztransib not only represents the first known intact Transib element in any organism, but has been recently transposed in H. zea. The fact that the intact Hztransib is transcribed into a 3′-truncated nonstop mRNA which may encode a non-functional transposase or be blocked from further translation via nonstop mRNA decay suggests that it is silenced at the translational or transpositional level.

A Genomic Screen in Yeast Reveals Novel Aspects of Nonstop mRNA Metabolism

Nonstop mRNA decay, a specific mRNA surveillance pathway, rapidly degrades transcripts that lack in-frame stop codons. The cytoplasmic exosome, a complex of 3'-5' exoribonucleases involved in RNA degradation and processing events, degrades nonstop transcripts. To further understand how nonstop mRNAs are recognized and degraded, we performed a genomewide screen for nonessential genes that are required for nonstop mRNA decay. We identified 16 genes that affect the expression of two different nonstop reporters. Most of these genes affected the stability of a nonstop mRNA reporter. Additionally, three mutations that affected nonstop gene expression without stabilizing nonstop mRNA levels implicated the proteasome. This finding not only suggested that the proteasome may degrade proteins encoded by nonstop mRNAs, but also supported previous observations that rapid decay of nonstop mRNAs cannot fully explain the lack of the encoded proteins. Further, we show that the proteasome and Ski7p affected expression of nonstop reporter genes independently of each other. In addition, our results implicate inositol 1,3,4,5,6-pentakisphosphate as an inhibitor of nonstop mRNA decay.

Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function

Cells routinely make mistakes. Some mistakes are encoded by the genome and may manifest as inherited or acquired diseases. Other mistakes occur because metabolic processes can be intrinsically inefficient or inaccurate. Consequently, cells have developed mechanisms to minimize the damage that would result if mistakes went unchecked. Here, we provide an overview of three quality control mechanisms--nonsense-mediated mRNA decay, nonstop mRNA decay, and no-go mRNA decay. Each surveys mRNAs during translation and degrades those mRNAs that direct aberrant protein synthesis. Along with other types of quality control that occur during the complex processes of mRNA biogenesis, these mRNA surveillance mechanisms help to ensure the integrity of protein-encoding gene expression.

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells

We investigated the fate of aberrant mRNAs lacking in-frame termination codons (called nonSTOP mRNA) in mammalian cells. We found that translation of nonSTOP mRNA was considerably repressed although a corresponding reduction of mRNA was not observed. The repression appears to be post-initiation since (i) repressed nonSTOP mRNAs were associated with polysomes, (ii) translation of IRES-initiated and uncapped nonSTOP mRNA were repressed, and (iii) protein production from nonSTOP mRNA associating with polysomes was significantly reduced when used to program an in vitro run-off translation assay. NonSTOP mRNAs distributed into lighter polysome fractions compared to control mRNAs encoding a stop codon, and a significant amount of heterogeneous polypeptides were produced during in vitro translation of nonSTOP RNAs, suggesting premature termination of ribosomes translating nonSTOP mRNA. Moreover, a run-off translation assay using hippuristanol and RNAse protection assays suggested the presence of a ribosome stalled at the 3' end of nonSTOP mRNAs. Taken together, these data indicate that ribosome stalling at the 3' end of nonSTOP mRNAs can block translation by preventing upstream translation events.

Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast

mRNA surveillance system represses the expression of nonstop mRNA by rapid mRNA degradation and translation repression. Here we show that the level of protein product of nonstop mRNA containing a poly(A) tail was reduced 100-fold, and this reduction was due to rapid mRNA degradation, translation repression, and protein destabilization, at least in part, by the proteasome. Insertion of a poly(A) tract upstream of a termination codon resulted in translation repression and protein destabilization, but not rapid mRNA decay. We propose that translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization.

RNase R degrades non‐stop mRNAs selectively in an SmpB‐tmRNA‐dependent manner

The SmpB-tmRNA-mediated trans-translation system has two well-established activities: rescuing ribosomes stalled on aberrant mRNAs and marking the associated protein fragments for proteolysis. Although the causative non-stop mRNAs are known to be degraded, little is known about the enabling mechanism or the RNases involved in their disposal. We report that Escherichia coli has an enabling mechanism that requires RNase R activity and is dependent on the presence of SmpB protein and tmRNA, suggesting a requirement for active transtranslation in facilitating RNase R engagement and promoting non-stop mRNA decay. Interestingly, this selective transcript degradation by RNase R targets aberrant (non-stop and multiple-rare-codon containing) mRNAs and does not affect the decay of related messages containing in-frame stop codons. Most surprisingly, RNase II and PNPase do not play a significant role in tmRNA-facilitated disposal of aberrant mRNAs. These findings demonstrate that RNase R is a crucial component of the trans-translation-mediated non-stop mRNA decay process, thus providing a requisite activity well suited to complement the ribosome rescue and protein tagging functions of this unique quality control system.

Reduced action of polypeptide release factors induces mRNA cleavage and tmRNA tagging at stop codons in Escherichia coli

Certain C-terminal sequences of nascent peptide cause an efficient protein tagging by tmRNA system at stop codons in Escherichia coli. Here, we demonstrate that both mRNA cleavage and tmRNA tagging occur at UAG stop codon recognized specifically by polypeptide release factor 1 (RF-1) when the activity of RF-1 is reduced by a mutation in the prfA gene without requirement of particular C-terminal sequences of nascent peptide. The tmRNA tagging and mRNA cleavage in the prfA mutant were eliminated when the wild-type RF-1 but not RF-2 was supplied from plasmid. In addition, depletion of either RF-1 or RF-2 induces endonucleolytic cleavage and tmRNA tagging at UAG or UGA stop codons respectively. We conclude that ribosome stalling at the cognate stop codon caused by reduced activity or expression of RF-1 or RF-2 is responsible for mRNA cleavage. The present data along with our previous studies strongly suggest that ribosome stalling leads to endonucleolytic cleavage of mRNA in general resulting in non-stop mRNA and that the 3' end of non-stop mRNA is probably only target for the tmRNA system.

TmRNA determinants required for facilitating nonstop mRNA decay

In bacteria, ribosomes stalled on nonstop mRNAs are rescued by tmRNA in a unique process called trans-translation. The two known tmRNA functions in trans-translation are (1) a tRNA-like function, which transfers the partially synthesized protein fragment to itself; and (2) an mRNA-like function, which enables ribosomes to resume and terminate translation on tmRNA as a surrogate template. We present evidence to demonstrate that tmRNA performs a third function, namely, facilitating the degradation of the causative defective mRNA. Our investigations have revealed the identity of key sequence determinants that promote the degradation of the nonstop mRNA. These sequence determinants are located in the distal part of the tmRNA open reading frame, encoding the ultimate, penultimate, and anti-penultimate amino acids of the peptide tag. We show that mutation of these tmRNA sequence elements has an adverse affect on the disposal of the nonstop mRNA, while leaving the tRNA and mRNA functions entirely unaffected. More significantly, specific mutations that change the nucleotide sequence of the peptide-reading frame without altering the nature or identity of the encoded amino acids still exhibit the characteristic defect in nonstop mRNA decay. In contrast, mutations in codons 3, 4, 5, and 6 of the tmRNA open reading frame do not have an adverse affect on degradation of defective mRNAs. Based on these results, we propose that tmRNA plays an important role in promoting the decay of nonstop mRNAs and that sequence elements in the distal segment of the peptide-reading frame make sequence-specific contributions that are crucial for this activity.

Plant mitochondrial genes can be expressed from mRNAs lacking stop codons

The mRNAs of the nad6 and ccmC genes of Arabidopsis and cauliflower were found to be processed upstream of the inframe stop codons. This result was confirmed by northern hybridization and by RT-PCR. There is no evidence that an alternative stop codon is created post-transcriptionally, either by RNA editing or by polyadenylation. The non-stop mRNAs are found in the high molecular weight polysomal fractions, suggesting that they are translated. Using antibodies directed against CcmC, the corresponding protein was detected in Arabidopsis mitochondrial extracts. These observations raise the question of how the plant mitochondrial translation system deals with non-stop mRNAs.