Jean-Pierre Rousset

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.

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 N6‐threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons. This was confirmed as a yeast mutant lacking Kae1 is devoid of t6A. t6A− strains were also used to reveal that t6A has a critical role in initiation codon restriction to AUG and in restricting frameshifting at tandem ANN codons. We also showed that YaeZ, a YgjD paralog, is required for YgjD function in vivo in bacteria. This work lays the foundation for understanding the pleiotropic role of this universal protein family.

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 Swi1 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. [PSI+] modifies cellular fitness and induces several phenotypes according to the genetic background. An elegant series of studies has demonstrated that several [PSI+]-induced phenotypes occur as a consequence of decreased translational termination efficiency. However, the genes whose expression levels are controlled by [PSI+] remain largely unknown. Here, we show that [PSI+] enhances expression of antizyme, a negative regulator of cellular polyamines, by modulating the +1 frameshifting required for its expression. Our study also demonstrates that [PSI+] greatly affects cellular polyamines in yeast. We show that modification of the cellular content of polyamines by the prion accounts for half of the [PSI+]-induced phenotypes. Antizyme is the first protein to be described for which expression in its functional form is stimulated by [PSI+].

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. Since, many studies have validated this approach, but despite the promise of PTC readthrough therapies, the mechanisms of translation termination remain to be precisely elucidated before even more progress can be made. Here, we review the molecular basis for PTC readthrough in eukaryotes and describe currently available compounds with significant therapeutic potential for treating genetic disorders and cancer.

UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals

BackgroundTranslation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.ResultsWe have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10-4. We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed.ConclusionsWe conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.

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.

Nonsense-mediated decay mutants do not affect programmed -1 frameshifting.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

Towards a computational model for −1 eukaryotic frameshifting sites

Abstract 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/ Contact: bekaert@igmors.u-psud.frjpforest@lri.frchris@lri.fr * To whom correspondence should be addressed.

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

Abstract 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 Ψ39 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.

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. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61–64) and YxCxxxF (amino acids 124–131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons.