Rab-GDI complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes.

In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium).

Sense from nonsense: therapies for premature stop codon diseases.

Ten percent of inherited diseases are caused by premature termination codon (PTC) mutations that lead to degradation of the mRNA template and to the production of a non-functional, truncated polypeptide. In addition, many acquired mutations in cancer introduce similar PTCs. In 1999, proof-of-concept for treating these disorders was obtained in a mouse model of muscular dystrophy, when administration of aminoglycosides restored protein translation by inducing the ribosome to bypass a PTC.

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.

[Allele-specific therapy: suppression of nonsense mutations by readthrough inducers].

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein.

Readthrough of premature termination codons in the adenomatous polyposis coli gene restores its biological activity in human cancer cells.

The APC tumor suppressor gene is frequently mutated in human colorectal cancer, with nonsense mutations accounting for 30% of all mutations in this gene. Reintroduction of the WT APC gene into cancer cells generally reduces tumorigenicity or induces apoptosis. In this study, we explored the possibility of using drugs to induce premature termination codon (PTC) readthrough (aminoglycosides, negamycin), as a means of reactivating endogenous APC.

A mutation within the C-terminal domain of Sup35p that affects [PSI+] prion propagation.

The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth.

A viable hypomorphic allele of the essential IMP3 gene reveals novel protein functions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion.

A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification.

The YgjD/Kae1 family (COG0533) has been on the top-10 list of universally conserved proteins of unknown function for over 5 years. It has been linked to DNA maintenance in bacteria and mitochondria and transcription regulation and telomere homeostasis in eukaryotes, but its actual function has never been found. Based on a comparative genomic and structural analysis, we predicted this family was involved in the biosynthesis of N(6)-threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons.

Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides.

Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein.

Translational errors: from yeast to new therapeutic targets.

Errors occur randomly and at low frequency during the translation of mRNA. However, such errors may also be programmed by the sequence and structure of the mRNA. These programmed events are called 'recoding' and are found mostly in viruses, in which they are usually essential for viral replication.

Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy.

Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2'-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains-the A-site finger and Helix 69 (six and five modifications).

The paradox of viable sup45 STOP mutations: a necessary equilibrium between translational readthrough, activity and stability of the protein.

The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q --> TAA, 242R --> TGA, 317L --> TAG).

Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1.

Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3-a GTPase protein-probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release.

Epigenetic control of polyamines by the prion [PSI+].

Prion proteins are found in mammals and yeast, and can transmit diseases and encode heritable phenotypic traits. In Saccharomyces cerevisiae, eRF3, Rnq1, Ure2 and Swil are functional proteins with a soluble conformation that can switch to a non-functional, amyloid conformation denoted as [PSI+], [PIN+], [URE3] and [SWI+], respectively. The prion [PSI+] corresponds to an aggregated conformation of the translational release factor eRF3, which suppresses nonsense codons.

Ribosome performance is enhanced by a rich cluster of pseudouridines in the A-site finger region of the large subunit.

The large subunit rRNA in eukaryotes contains an unusually dense cluster of 8-10 pseudouridine (Psi) modifications located in a three-helix structure (H37-H39) implicated in several functions. This region is dominated by a long flexible helix (H38) known as the "A-site finger" (ASF). The ASF protrudes from the large subunit just above the A-site of tRNA binding, interacts with 5 S rRNA and tRNA, and through the terminal loop, forms a bridge (B1a) with the small subunit.

A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability.

Background: When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved.

Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes.

Background: The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough.

Adding pyrrolysine to the Escherichia coli genetic code.

Pyrrolysyl-tRNA synthetase and its cognate suppressor tRNA(Pyl) mediate pyrrolysine (Pyl) insertion at in frame UAG codons. The presence of an RNA hairpin structure named Pyl insertion structure (PYLIS) downstream of the suppression site has been shown to stimulate the insertion of Pyl in archaea. We study here the impact of the presence of PYLIS on the level of Pyl and the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc) incorporation using a quantitative lacZ-luc tandem reporter system in an Escherichia coli context.

Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors.

In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universal-code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition.

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.

In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study.

Background: Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel activated by cyclic AMP (cAMP). The most frequent mutation found in 70% of CF patients is F508del, while premature stop mutations are found in about 10% of patients. In vitro aminoglycoside antibiotics (e.

Identification of programmed translational -1 frameshifting sites in the genome of Saccharomyces cerevisiae.

Frameshifting is a recoding event that allows the expression of two polypeptides from the same mRNA molecule. Most recoding events described so far are used by viruses and transposons to express their replicase protein. The very few number of cellular proteins known to be expressed by a -1 ribosomal frameshifting has been identified by chance.