Comparative analysis of base biases around the stop codons in six eukaryotes

Using full-length cDNA sequences, a comparative analysis of sequence patterns around the stop codons in six eukaryotes was performed. Here, it was showed that the codon immediately before and after the stop codons (defined as -1 codon and +1 codon, respectively) were much more biased than other examined positions, especially at the second position of -1 codons and the first position of +1 codons which were rich in As/Us and purines, respectively, for most species. The author speculated that strongly biased sequence pattern from position -2 to +4 might act as an extended translation termination signal. Translation termination was catalyzed by release factors that recognized the stop codons. The multiple amino acid sequence alignment of eukaryotic release factor 1 (eRF1) of 20 species showed that there were 16 residue sites that were strictly conserved, especially the invariant amino acids Ile70 and Lys71. Accordingly, it could be inferred that those candidate amino acids might involve in the recognition process. Moreover, the possible stop signal recognition hypothesis was also discussed herein.     

Analysis of sequence patterns proximal to the stop codons in Oryza sativa

Abstract

A comprehensive analysis of sequence patterns around the stop codons was performed, by using more than 26,000 rice full-length cDNA sequences. Here it is shown that the bias was most outstanding at the position immediately before the stop codons (?1 codon), where the AAC codon was strongly preferred among ANC codons. Compared with other positions, the codon immediately after the stop codons (+1 codon) also displayed an apparent difference, and had a strong consensus for base A at the first, C at the second, and A at the third letters, respectively. Notably, the base biases at the positions directly downstream of the stop codons, such as the +4, +5 and +6 positions, were much stronger than other positions in the 3′-UTR region, suggesting that those base positions might act as an extended stop signal in the process of protein synthesis. Examination of the relationship between sequence pattern and gene expression level, assessed by CAI values and EST counting, revealed a tendency towards bigger base biases for highly expressed genes. It could be inferred that the translation stop signal is possibly involved in many sequence recognition elements other than the stop codons; highly expressed genes should hold strong sequence consensus around the stop codons for efficient translation termination.     

Computational analysis of stop codon readthrough in D.melanogaster

MOTIVATION: Readthrough is an unusual process in which a stop codon is misread or skipped. Recently it has been shown that some translation is regulated by the readthrough reactions although the complete mechanism is not clear. Therefore, the discovery of 'readthrough genes' is important for further investigation of their cellular roles, which may provide additional insights into the mechanism of translational regulation. RESULTS: We constructed a system that lists candidates of readthrough genes based on the existence of a 'protein motif' at the 3' untranslated region (UTR). Using this system, we extracted 85 candidates from 4082 nucleic acid sequences of Drosophila melanogaster in GenBank database. The sequences of these candidates had a slightly more stable secondary structure and different base preferences compared to the non-candidates. As these features are known to have an effect on readthrough events, we would like to suggest that these candidates contain actual readthrough genes. AVAILABILITY: Source code of the system is available upon request.     

Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the -1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.     

The C-terminal amino acid sequence of nascent peptide is a major determinant of SsrA tagging at all three stop codons

Recent studies on endogenous SsrA-tagged proteins have revealed that the tagging could occur at a position corresponding to the normal termination codon. During the study of SsrA-mediated Lacl tagging (Abo et al., EMBO J, 2000 19:3762-3769), we found that a variant Lacl (Lacl deltaC1) lacking the last C-terminal amino acid residue is efficiently tagged in a stop codon-dependent manner. SsrA tagging of Lacl deltaC1 occurred efficiently without Lacl binding to the lac operators at any one of three stop codons. The C-terminal (R)LESG peptide of Lacl deltaC1 was shown to trigger the SsrA tagging of an unrelated protein (CRP) when fused to its C terminus. Mass spectrometry analysis of the purified fusion proteins revealed that SsrA tagging occurs at a position corresponding to the termination codon. The alteration of the amino acid sequence but not the nucleotide sequence of the C-terminal portion eliminated the tagging. We also showed that the tagging-provoking sequences cause an efficient translational readthrough at UGA but not UAA codons. In addition, we found that C-terminal dipeptides known to induce an efficient translation readthrough could cause an efficient tagging at stop codons. We conclude that the amino acid sequence of nascent polypeptide prior to stop codons is a major determinant for the SsrA tagging at all three stop codons.     

Comprehensive sequence analysis of translation termination sites in various eukaryotes

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.     

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.     

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.     

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.     

C-domain of translation termination factor eRF1 neighbors stop codon at the 80S ribosomal A site

Positioning of stop codon and the adjacent triplet downstream of it with respect to the components of human 80S termination complex was studied with the use of mRNA analogues that bore stop signal UPuPuPu (Pu is A or G) and photoactivatable perfluoroaryl azide group. This group was attached to one of nucleotides of the stop signal or 3' of it (in positions +4 to +9 with respect to the first nucleotide of the P site codon). It was shown that upon mild UV irradiation the mRNA analogues crosslinked to components of model complexes imitating state of 80S ribosome in the course of translation termination. It was found that termination factors eRF1 and eRF3 do not affect mutual arrangement of stop signal and the 18S rRNA. Factor eRF1 was shown to cross-link to modified nucleotides in positions +5 to +9 (ability of eRF1 to cross-link to stop codon nucleotide in position +4 was shown earlier). Fragments of eRF1 containing cross-linking sites of the mRNA analogues were determined. In fragment 52-195 (containing the N-domain and a part of the M-domain) we have found cross-linking sites of the analogues that bore modifying groups on A or G in positions +5 to +9 or at the terminal phosphate of nucleotide in position +7. For mRNA analogues bearing modifying groups on G site of cross-linking from positions +5 to +7 was found in the eRF1 fragment     

Fine-tuning of translation termination efficiency in Saccharomyces cerevisiae involves two factors in close proximity to the exit tunnel of the ribosome

In eukaryotes, release factors 1 and 3 (eRF1 and eRF3) are recruited to promote translation termination when a stop codon on the mRNA enters at the ribosomal A-site. However, their overexpression increases termination efficiency only moderately, suggesting that other factors might be involved in the termination process. To determine such unknown components, we performed a genetic screen in Saccharomyces cerevisiae that identified genes increasing termination efficiency when overexpressed. For this purpose, we constructed a dedicated reporter strain in which a leaky stop codon is inserted into the chromosomal copy of the ade2 gene. Twenty-five antisuppressor candidates were identified and characterized for their impact on readthrough. Among them, SSB1 and snR18, two factors close to the exit tunnel of the ribosome, directed the strongest antisuppression effects when overexpressed, showing that they may be involved in fine-tuning of the translation termination level.     

Tandem termination signal in plant mRNAs

It was proposed that if some mRNA characteristics resulted in a low efficiency of termination signal, an additional closely located stop codon (tandem stop codons) could be used to prevent the harmful readthrough. However, the role of tandem terminators in higher eukaryotes was not verified and remains hypothetical. In this work the sequence features of Arabidopsis thaliana and Oryza sativa mRNAs were analyzed. It was found that plant mRNAs with UGA terminator were characterized by a higher frequency of nonsense codons in the first triplet position of 3′-UTR that could result from a weak natural selection for “reserve” stop signal. Interestingly, the presence of tandem stop codons positively correlated with a specific amino acid composition in the C-terminal position of the encoded proteins. In particular, C-terminal glycine positively correlated with significantly higher frequencies of reserve terminators at the beginning positions of 3′-UTR in UGA-containing mRNAs. This finding coincides with some earlier observations concerning the role of glycine and its codons in inefficient termination of translation and recoding (e.g., 2A oligopeptide).     

Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae

In-frame stop codons normally signal termination during mRNA translation, but they can be read as ‘sense’ (readthrough) depending on their context, comprising the 6 nt preceding and following the stop codon. To identify novel contexts directing readthrough, under-represented 5′ and 3′ stop codon contexts from Saccharomyces cerevisiae were identified by genome-wide survey in silico. In contrast with the nucleotide bias 3′ of the stop codon, codon bias in the two codon positions 5′ of the termination codon showed no correlation with known effects on stop codon readthrough. However, individually, poor 5′ and 3′ context elements were equally as effective in promoting stop codon readthrough in vivo, readthrough which in both cases responded identically to changes in release factor concentration. A novel method analysing specific nucleotide combinations in the 3′ context region revealed positions +1,2,3,5 and +1,2,3,6 after the stop codon were most predictive of termination efficiency. Downstream of yeast open reading frames (ORFs), further in-frame stop codons were significantly over-represented at the +1, +2 and +3 codon positions after the ORF, acting to limit readthrough. Thus selection against stop codon readthrough is a dominant force acting on 3′, but not on 5′, nucleotides, with detectable selection on nucleotides as far downstream as +6 nucleotides. The approaches described can be employed to define potential readthrough contexts for any genome.     

Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.     

Ribosomal Readthrough at a Short UGA Stop Codon Context Triggers Dual Localization of Metabolic Enzymes in Fungi and Animals

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.     

The effect of eukaryotic release factor depletion on translation termination in human cell lines

Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome. Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide. Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension. The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs. Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines. Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells. The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems. We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells. These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner.     

Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast

The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3.     

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.     

Gene overexpression as a tool for identifying new trans-acting factors involved in translation termination in Saccharomyces cerevisiae

In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.