Biomedical subjects

Jean-Pierre Rousset

Publications and source records attributed to Jean-Pierre Rousset.

9 recordsLinked to original sources

PubMed2005

An extended signal involved in eukaryotic -1 frameshifting operates through modification of the E site tRNA.

By using a sensitive search program based on hidden Markov models (HMM), we identified 74 viruses carrying frameshift sites among 1500 fully sequenced virus genomes. These viruses are clustered in specific families or genera. Sequence analysis of the frameshift sites identified here, along with previously characterized sites, identified a strong bias toward the two nucleotides 5' of the shifty heptamer signal. Functional analysis in the yeast Saccharomyces cerevisiae demonstrated that high frameshifting efficiency is correlated with the presence of a Psi39 modification in the tRNA present in the E site of the ribosome at the time of frameshifting. These results demonstrate that an extended signal is involved in eukaryotic frameshifting and suggest additional interactions between tRNAs and the ribosome during decoding.

Base Sequence↗

PubMed2004

Reprogrammed genetic decoding in cellular gene expression.

Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes.

Animals↗

PubMed2004

The major 5' determinant in stop codon read-through involves two adjacent adenines.

The aim of this approach was to identify the major determinants, located at the 5' end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae. We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene. Variations at these positions modulated translational read-through efficiency approximately 16-fold. The major effect was imposed by the two nucleotides immediately upstream of the stop codon. We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site. We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5' determinant of read-through efficiency.

Adenine Nucleotides↗

PubMed2003

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.

Base Sequence↗

PubMed2003

Towards a computational model for -1 eukaryotic frameshifting sites.

MOTIVATION: Unconventional decoding events are now well acknowledged, but not yet well formalized. In this study, we present a bioinformatics analysis of eukaryotic -1 frameshifting, in order to model this event. RESULTS: A consensus model has already been established for -1 frameshifting sites. Our purpose here is to provide new constraints which make the model more precise. We show how a machine learning approach can be used to refine the current model. We identify new properties that may be involved in frameshifting. Each of the properties found was experimentally validated. Initially, we identify features of the overall model that are to be simultaneously satisfied. We then focus on the following two components: the spacer and the slippery sequence. As a main result, we point out that the identity of the primary structure of the so-called spacer is of great importance. AVAILABILITY: Sequences of the oligonucleotides in the functional tests are available at http://www.igmors.u-psud.fr/rousset/bioinformatics/.

Algorithms↗

PubMed2002

Up-regulation of tRNA biosynthesis affects translational readthrough in maf1-delta mutant of Saccharomyces cerevisiae.

Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-delta mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ- luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-delta compared with the parental strain. The maf1-delta mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1- Delta using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11or by [PSI(+)], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1- Delta increase translational fidelity and, independently, affect growth under special conditions.

Codon, Nonsense↗

PubMed2002

Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency.

Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.

Anticodon↗

PubMed2002

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.

Codon, Terminator↗

PubMed2002

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.

Cytoskeleton↗