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.     

Deciphering the molecular mechanism of stop codon readthrough

Recognition of the stop codon by the translation machinery is essential to terminating translation at the right position and to synthesizing a protein of the correct size. Under certain conditions, the stop codon can be recognized as a coding codon promoting translation, which then terminates at a later stop codon. This event, called stop codon readthrough, occurs either by error, due to a dedicated regulatory environment leading to generation of different protein isoforms, or through the action of a readthrough compound. This review focuses on the mechanisms of stop codon readthrough, the nucleotide and protein environments that facilitate or inhibit it, and the therapeutic interest of stop codon readthrough in the treatment of genetic diseases caused by nonsense mutations.     

How translation termination factor eRF1 Euplotes does not recognise UGA stop codon

In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.     

Identification of stop codon readthrough genes in Saccharomyces cerevisiae

We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon. For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs). Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25%. For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA. Using isogenic [PSI⁺] and [psi^–] yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough. However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism. Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene. This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.     

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.     

Polyamines enhance readthrough of the UGA termination codon in a mammalian messenger RNA

The polyamines spermidine and spermine stimulate the readthrough of the UGA termination codon of rabbit beta-globin mRNA when it is translated in a rabbit reticulocyte cell-free system. The other major polyamine, putrescine, does not show this effect. The polyamine induced readthrough is specific for UGA as the UAA termination codon of alpha-globin mRNA is not read through and general translational misreading errors are not occurring in the presence of spermidine or spermine. The probable mechanism of this effect and some possible regulatory implications are discussed.     

Evidence of efficient stop codon readthrough in four mammalian genes

Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5′ and 3′ of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.     

Growth at low temperature suppresses readthrough of the UGA stop codon during the expression of Bacillus subtilis flgM gene in Escherichia coli

The efficient production of recombinant proteins in Escherichia coli requires a proper termination of translation to ensure the synthesis of only the desired product. During the recombinant production of Bacillus subtilis flgM in E. coli, we detected an additional polypeptide of molecular mass higher than the expected, corresponding to a product of a translational readthrough of the UGA stop codon. In this paper we show that the readthrough was abolished when the synthesis of the recombinant protein was carried out at 25 degrees C. The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed.     

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.     

Aminoglycosides and other factors promoting stop codon readthrough in human cells

Enhanced stop codon readthrough is a potential treatment strategy for diseases caused by nonsense mutations. Here, we compare readthrough levels induced by three types of factors: aminoglycoside antibiotics, suppressor tRNAs, and factors decreasing translation termination efficiency. We show that the highest levels of readthrough were obtained by prolonged treatment with aminoglycosides and suppressor tRNAs, whereas prolonged depletion of release factors induced only a moderate increase in readthrough. We discuss the benefits and inconvenients of the three types of factors for their use in the therapy of diseases caused by premature stop codons.     

Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.     

Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae

The efficiency of translation termination in yeast can vary several 100-fold, depending on the context around the stop codon. We performed a computer analysis designed to identify yeast open reading frames (ORFs) containing a readthrough motif surrounding the termination codon. Eight ORFs were found to display inefficient stop codon recognition, one of which, PDE2, encodes the high-affinity cAMP phosphodiesterase. We demonstrate that Pde2p stability is very impaired by the readthrough-dependent extension of the protein. A 20-fold increase in readthrough of PDE2 was observed in a [PSI+] as compared with a [psi-] strain. Consistent with this observation, an important increase in cAMP concentration was observed in suppressor backgrounds. These results provide a molecular explanation for at least some of the secondary phenotypes associated with suppressor backgrounds.     

Computational identification and sequence analysis of stop codon readthrough genes in Oryza sativa

Using an approach based on the Readthrough Candidate Extraction System (RCES), we extracted 111 candidates from 9620 gene sequences of rice. The results of homology search and sequence analysis demonstrated that these candidates included actual readthrough genes that would be important for further investigating the mechanism of translation termination regulated by readthrough event, and could also give some useful clues for functional genome annotation. Between the candidates and non-candidates of gene sequences in rice, there exist significant base biases at the positions surrounding the stop codons. These positions, especially both -1 and +4, are referred to as part of an extended stop signal. In candidates, G at position -1, and G or C at position +4 are much more favored than that in non-candidates. Both stop sequence patterns, GUAGC and GUGAG, might drive high readthrough efficiency in rice. Secondary structure analysis revealed that the -1 and +1 amino acids around the first stop codon of candidates have a strong bias toward arginine, particularly the +1 position (20.7%), which indicated that the amino acids at the readthrough region being frequently located in the hydrophilic region of beta-turn might be a determinant for efficient translation termination or not.     

A novel stop codon readthrough mechanism produces functional Headcase protein in Drosophila trachea

Translational regulation provides an efficient means to control the localization and production of proteins. The headcase (hdc) mRNA in Drosophila generates two overlapping proteins as a result of translational readthrough of an internal UAA stop codon. This readthrough event is necessary for the function of hdc as a branching inhibitor during tracheal development. By ectopic expression of different Hdc proteins in the trachea, we show that the long Hdc form alone, can function as a potent branching inhibitor whose activity is proportional to its amount. The suppression of termination in the hdc mRNA is not stop-codon dependent, suggesting that the readthrough does not involve codon specific suppressors. We have identified an 80 nucleotide sequence immediately downstream of the UAA, which is necessary and sufficient to confer termination readthrough in a heterologous mRNA. We present a novel mechanism of eukaryotic translational termination suppression that may regulate the amount of functional Hdc.     

Identification and functional characterization of mRNAs that exhibit stop codon readthrough in Arabidopsis thaliana

Stop codon readthrough (SCR) is the process of continuation of translation beyond the stop codon, generating protein isoforms with C-terminal extensions. SCR has been observed in viruses, fungi, and multicellular organisms, including mammals. However, SCR is largely unexplored in plants. In this study, we have analyzed ribosome profiling datasets to identify mRNAs that exhibit SCR in Arabidopsis thaliana. Analyses of the ribosome density, ribosome coverage, and three-nucleotide periodicity of the ribosome profiling reads in the mRNA region downstream of the stop codon provided strong evidence for SCR in mRNAs of 144 genes. We show that SCR generated putative evolutionarily conserved nuclear localization signals, transmembrane helices, and intrinsically disordered regions in the C-terminal extensions of several of these proteins. Furthermore, gene ontology functional enrichment analysis revealed that these 144 genes belong to three major functional groups—translation, photosynthesis, and abiotic stress tolerance. Using a luminescence-based readthrough assay, we experimentally demonstrated SCR in representative mRNAs belonging to each of these functional classes. Finally, using microscopy, we show that the SCR product of one gene that contains a nuclear localization signal at the C-terminal extension, CURT1B, localizes to the nucleus as predicted. Based on these observations, we propose that SCR plays an important role in plant physiology by regulating protein localization and function.     

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.     

Dietary selenium deficiency and supplementation differentially modulate the expression of two ER-resident selenoproteins (selenoprotein K and selenoprotein M) in the ovaries of aged mice: Preliminary data

In the present report, we determined the impact of dietary selenium (Se) deficiency and supplementation on the expression of two ER-resident selenoproteins i.e., Selenok and Selenom in the ovaries of aging mice. The mRNA expression of Selenok and Selenom (RT-qPCR) was significantly higher in the ovaries of mice fed diets supplemented with inorganic (ISe-S: 0.33 mg Se/kg) and organic (OSe-S: 0.33 mg Se/kg) Se compared to those fed a Se-deficient (Se-D: 0.08 mg Se/kg) diet and both Se-adequate (ISe-A: 0.15 mg Se/kg and OSe-A: 0.15 mg Se/kg) diets. Similarly, the protein signals of SELENOK (immunofluorescence assay) were also significantly higher in the Se-supplemented groups compared to those fed Se-D and Se-adequate (ISe-A and OSe-A) diets. Meanwhile, the rate of in vitro-produced blastocysts developing from MII oocytes was also evaluated and it was revealed that this rate was significantly higher in the Se-supplemented mice compared to those fed a Se-D diet. Altogether, the dietary Se supplementation increased the expression of Selenok (also its protein expression) and Selenom in the ovaries of aging mice, potentially contributing to an improved developmental potential of in vitro-matured M II oocytes.     

哺乳动物硒蛋白的研究进展

硒是哺乳动物和人必需的微是元素。硒的生物学功能主要是以硒蛋白的形式表现的。到目前为止,已经克隆并测定ｃＤＮＡ顺序的哺乳动物硒蛋白有９种停,它们是细胞内谷胱甘肽过氧化物酶、细胞外谷胱甘肽过氧化物酶、磷脂氢谷胱甘肽过氧化物酶、胃肠谷胱甘肽过氧化物酶、Ｉ型碘化甲状腺原氨酸５′脱碘酶、Ⅱ型碘化甲状腺原氨酸５′脱磺酶、Ⅲ型碘化甲状腺原氨酸５′脱碘酶、硒蛋白Ｐ和硒蛋白Ｗ。这些硒蛋白中硒参入到蛋白分子是通过硒半