Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.     

CRISPR-free,programmable RNA pseudouridylation to suppress premature termination codons

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Localization of initiating codons in RNA prokaryotes messengers by learning technics

Learning processes are applied to the recognition of protein coding regions in prokaryotes. Non-contradictory, statistical rules are deduced from a set of known examples of coding regions. These rules allow us to build characteristic patterns on the m-RNA upstream the initiating codon. These rules are applied to recognize more than 180 coding sequences.     

Functional importance of RNA interactions in selection of translation initiation codons

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA–small ribosomal subunit–initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis -acting mRNA elements and trans -acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.     

CUG codons inCandida spp.

Codon CUG is used for serine instead of for leucine, its usual assignment, in several yeasts of the genusCandida. We propose a series of steps for the reassignment, including disappearance of leucine CUG and its anticodon CAG, formation of a new serine tRNA, with anticodon CAG, from a duplication of the gene for serine tRNA (IGA), and then production of CUG codons by mutation at sites that are mostly nonessential.     

When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells.

It appears that no organism is immune to the effects of nonsense codons on mRNA abundance. The study of how nonsense codons alter RNA metabolism is still at an early stage, and our current understanding derives more from incidental vignettes than from experimental undertakings that address molecular mechanisms. Challenges for the future include identifying the gene products and RNA sequences that function in nonsense mediated RNA loss, resolving the cause and consequences of there apparently being more than one cellular site and mechanism for nonsense-mediated RNA loss, and understanding how these sites and mechanisms are related to both constitutive and specialized pathways of pre-mRNA processing and mRNA decay.     

TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus.

The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.     

Rare codons in E. coli and S. typhimurium signal sequences

Codon usage has been examined in the signal sequences of 27 genes encoding proteins which possess leader peptides, and are inner-membrane located or exported. The results have been compared with codon usage in the corresponding coding sequences of most of the mature proteins. A bias is observed in the usage of rare codons for two of the three hydrophobic amino acids for which there are rare codons. Since hydrophobic residues are predominant in leader peptides, we suggest that a resulting concentration of rare codons in the signal sequence may play a role (or have played a role in the evolutionary past) in the secretion process by delaying translation.     

Rare codons cluster

Most amino acids are encoded by more than one codon. These synonymous codons are not used with equal frequency: in every organism, some codons are used more commonly, while others are more rare. Though the encoded protein sequence is identical, selective pressures favor more common codons for enhanced translation speed and fidelity. However, rare codons persist, presumably due to neutral drift. Here, we determine whether other, unknown factors, beyond neutral drift, affect the selection and/or distribution of rare codons. We have developed a novel algorithm that evaluates the relative rareness of a nucleotide sequence used to produce a given protein sequence. We show that rare codons, rather than being randomly scattered across genes, often occur in large clusters. These clusters occur in numerous eukaryotic and prokaryotic genomes, and are not confined to unusual or rarely expressed genes: many highly expressed genes, including genes for ribosomal proteins, contain rare codon clusters. A rare codon cluster can impede ribosome translation of the rare codon sequence. These results indicate additional selective pressures govern the use of synonymous codons, and specifically that local pauses in translation can be beneficial for protein biogenesis.     

A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus

Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3' UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3' UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3' UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.     

Ribosome bypassing at serine codons as a test of the model of selective transfer RNA charging

Recently, a model of the flux of amino acids through transfer RNAs (tRNAs) and into protein has been developed. The model predicts that the charging level of different isoacceptors carrying the same amino acid respond very differently to variation in supply of the amino acid or of the rate of charging. It has also been shown that ribosome bypassing is specifically stimulated at 'hungry' codons calling for an aminoacyl-tRNA in short supply. We have constructed two reporters of bypassing, which differ only in the identity of the serine codon subjected to starvation. The stimulation of bypassing as a function of starvation differed greatly between the two serine codons, in good agreement with the quantitative predictions of the model.     

Determination of mutation trend in hemagglutinins by means of translation probability between RNA codons and mutated amino acids

In this study, we used the 183 translation probabilities between RNA codons and mutated amino acids to construct the theoretical distributions of mutated amino acids in hemagglutinins of influenza A virus. We then compared the actual distributions of mutated amino acids from 953 hemagglutinins with their theoretical ones. The results demonstrated that mutated amino acids generally follow the direction of the theoretical distributions governed by RNA codons. This, in turn, highlights the mutation trend of amino acids in hemagglutinins and provides a method for estimating possible mutations in a protein according to its theoretical distributions of mutated amino acids.     

Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.