Olivier Namy

• PhD
• Research Director at French National Centre for Scientific Research

Significance Protein translation is a key cellular process in which each codon of mRNAs has to be accurately and efficiently recognized by cognate tRNAs of a large repertoire of noncognate tRNAs. A successful decoding process is largely dependent on the presence of modified nucleotides within the anticodon loop, especially of tRNAs having to read A...

Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumour suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic...

Coding sequences are defined between a start and a stop codon. The appearance of a nonsense mutation in a coding sequence creates a premature termination codon (PTC), preventing the correct production of the corresponding protein by interrupting translation and inducing the degradation of the transcript via the nonsense‐mediated mRNA decay (NMD) pa...

Ribosome profiling is an emerging approach using deep sequencing of the mRNA part protected by the ribosome to study protein synthesis at the genome scale. This approach provides new insights into gene regulation at the translational level. In this review we describe the protocol to prepare polysomes and extract ribosome protected fragments before...

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the ident...

The N6-methyladenosine (m6A) modification of HIV-1 RNAs plays an essential role in regulating viral infection. This modification has been widely studied but the number and precise positions of the m6A sites remain unclear due to the lack of precision of detection methods. We used the latest Nanopore chemistry and direct m6A base-calling option to i...

The development of ribosomal profiling (Riboseq) revealed the immense coding capacity of human and viral genomes. Here, we used Riboseq to delineate the translatome of HIV-1 in infected CD4⁺ T cells. In addition to canonical viral protein coding sequences (CDSs), we identify 98 alternative open reading frames (ARFs), corresponding to small Open Rea...

Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus that relies on alternative splicing, translational, and post-translational mechanisms to produce over 15 functional proteins from its single ~10 kb transcriptional unit. Using ribosome profiling, nascent protein labeling, RNA sequencing, and whole-proteomics of infected CD4 + T lym...

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q34 in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the ove...

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q34 in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the ove...

Epithelial–mesenchymal transition (EMT) involves profound changes in cell morphology, driven by transcriptional and epigenetic reprogramming. However, evidence suggests that translation and ribosome composition also play key roles in establishing pathophysiological phenotypes. Using genome-wide analyses, we reported significant rearrangement of the...

The development of ribosomal profiling (Riboseq) revealed the immense coding capacity of human and viral genomes. Here, we used Riboseq to delineate the first translatome of HIV-1 in infected CD4+ T cells. In addition to canonical viral protein coding sequences (CDSs), we identify 98 alternative open reading frames (ARFs), corresponding to small Op...

Background Pervasive translation is a widespread phenomenon that plays a critical role in the emergence of novel microproteins, but the diversity of translation patterns contributing to their generation remains unclear. Based on 54 ribosome profiling (Ribo-Seq) datasets, we investigated the yeast Ribo-Seq landscape using a representation framework...

Cellular processes are governed by the regulation of gene expression, often at the level of translation control. The mechanisms of control have been shown to operate at various levels, but there is growing evidence to suggest that rRNA modification patterns play a key role in driving translational modulation of the ribosome. We investigated the int...

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the overa...

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the overa...

Despite being predicted to lack coding potential, cytoplasmic long non-coding (lnc)RNAs can associate with ribosomes. However, the landscape and biological relevance of lncRNAs translation remains poorly studied. In yeast, cytoplasmic Xrn1-sensitive lncRNAs (XUTs) are targeted by the Nonsense-Mediated mRNA Decay (NMD), suggesting a translation-depe...

Around 12% of multiple myeloma (MM) cases harbour mutations in DIS3 , which encodes an RNA decay enzyme that controls the turnover of some long noncoding RNAs (lncRNAs). Although lncRNAs, by definition, do not encode proteins, some can be a source of (poly)peptides with biological importance, such as antigens. The extent and activities of these “co...

Some of the long noncoding (lnc)RNAs harbor potential to produce functional micro peptides. Despite their increasing significance, the regulatory dynamics of cytoplasmic lncRNA expression, decay, and translation remain poorly understood. Here, we investigate the role of ribonucleases in controlling cytoplasmic levels of lncRNAs. We observed large a...

Some long noncoding (lnc)RNAs harbor the potential to produce functional micropeptides. Despite the increasing recognition of their significance, the regulatory dynamics of cytoplasmic lncRNA expression, decay, and translation remain poorly understood. Here, we investigate the role of ribonucleases in controlling cytoplasmic levels of lncRNAs. By t...

Pervasive translation is a widespread phenomenon that plays an important role in de novo gene birth; however, its underlying mechanisms remain unclear. Based on multiple Ribosome Profiling (Ribo-Seq) datasets, we investigated the RiboSeq landscape of coding and noncoding regions of yeast. Therefore, we developed a representation framework which all...

Nonsense mutations account for 12 % of the cases of Cystic fibrosis (CF). The gene inactivation resulting from premature termination codon (PTC) can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. We recently identified a new readthrough inducer named TLN468, acting more effi...

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anti-codon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the over...

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medica...

Despite predicted to lack coding potential, cytoplasmic long non-coding (lnc)RNAs can associate with ribosomes, resulting in some cases into the production of functional peptides. However, the biological and mechanistic relevance of this pervasive lncRNAs translation remains poorly studied. In yeast, cytoplasmic Xrn1-sensitive lncRNAs (XUTs) are ta...

Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus which relies on alternative splicing, translational and post-translational mechanisms to produce more than 15 functional proteins from its single ∼10kb transcriptional unit. Here, we have applied ribosome profiling and nascent protein labeling at different time points during infect...

The noncoding genome plays an important role in de novo gene birth and in the emergence of genetic novelty. Nevertheless, how noncoding sequences’ properties could promote the birth of novel genes and shape the evolution and the structural diversity of proteins remains unclear. Therefore, by combining different bioinformatic approaches, we characte...

Premature termination codons (PTCs) account for 10% to 20% of genetic diseases in humans. The gene inactivation resulting from PTC can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medica...

RNA modifications play an essential role in determining RNA fate. Recent studies have revealed the effects of such modifications on all steps of RNA metabolism. These modifications range from the addition of simple groups, such as methyl groups, to the addition of highly complex structures, such as sugars. Their consequences for translation fidelit...

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning....

Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination...

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a pre...

Ribosome profiling (RiboSeq) has emerged as a powerful technique for studying the genome-wide regulation of translation in various cells. Several steps in the biological protocol have been improved, but the bioinformatics part of RiboSeq suffers from a lack of standardization, preventing the straightforward and complete reproduction of published re...

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favor the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selectio...

The noncoding genome plays an important role in de novo gene birth and in the emergence of genetic novelty. Nevertheless, how noncoding sequences' properties could promote the birth of novel genes and shape the evolution and the structural diversity of proteins remains unclear. Therefore, by combining different bioinformatic approaches, we characte...

Background & aims: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six...

Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination...

In addition to its role in translation termination, eRF3A has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with UPF1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbour upstream open reading frames in their 5ʹ...

Little is known about the rate of emergence of de novo genes, what their initial properties are, and how they spread in populations. We examined wild yeast populations (Saccharomyces paradoxus) to characterize the diversity and turnover of intergenic ORFs over short evolutionary timescales. We find that hundreds of intergenic ORFs show translation...

iGEM (pour international genetically engineered machine) est un concours international autour de la biologie synthétique réunissant des étudiants de toutes disciplines (mathématiques, physique, biologie, arts, etc.). « L’objectif est de construire un système biologique fonctionnel complexe, en assemblant des composants individuels moléculaires simp...

Messenger RNA translation is a complex process that is still poorly understood in eukaryotic or-ganelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view...

Introduction Metastasis of cancer cells, a primary cause of cancer death and a multiple and intricate processes, may complicate the clinical management and lead to a poor prognosis for cancer patients and has tremendous physical or economic impact to patients or communities. Pentraxin 3 (PTX3) plays an important role in innate immune responses and...

Little is known about the rate of emergence of genes de novo , how they spread in populations and what their initial properties are. We examined wild Saccharomyces paradoxus populations to characterize the diversity and turnover of intergenic ORFs over short evolutionary time-scales. We identified ~34,000 intergenic ORFs per individual genome for a...

Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount...

Significance Translational control is a cornerstone of gene-expression regulation in physiological and pathological contexts. The contribution of nonribosomal factors, including messenger RNAs (mRNAs) and mRNA-bound factors, to translational control have been extensively studied. Recently, the hypothesis of a ribosome-mediated regulation emerged, w...

Protein synthesis is a complex multi-step process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics....

The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not...

Ribosome profiling provides genome-wide information about translational regulation. However, there is currently no standard tool for the qualitative analysis of Ribo-seq data. We present here RiboTools, a Galaxy toolbox for the analysis of ribosome profiling (Ribo-seq) data. It can be used to detect translational ambiguities, stop codon readthrough...

In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be imp...

Threonylcarbamoyladenosine (t6A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t6A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t6A ma...

Prions are infectious proteins that can adopt a structural conformation that is then propagated among other molecules of the same protein. [PSI(+)] is an aggregated conformation of the translational release factor eRF3. [PSI(+)] modifies cellular fitness, inducing various phenotypes depending on genetic background. However, the genes displaying [PS...

poster présenté par Nathalie Barbier

oral présenté par N. Westbrook

communication orale présentée par Karen Perronet

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 modificat...

poster présenté par Nathalie Westbrook

poster présenté par Nathalie Westbrook

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 mo...

poster présenté conjointement par Hélène Chommy et Karen Perronet

Résumé : Nous présentons une étude de la durée de vie avant photoblanchiment du marqueur fluorescent ATTO647N à l'échelle de la molécule unique par microscopie de fluorescence en réflexion totale. Nous avons mesuré la durée de vie avant photoblanchiment dans un tampon usuel et dans des extraits cellulaires. Nous avons ensuite étudié l'influence de...

communication orale de Nicolas Fiszman

Présentation orale d'Antoine Le Gall

The [PSI (+) ] determinant in Saccharomyces cerevisiae is the prion protein corresponding to the eRF3 translation termination factor. Numerous infectious proteins have been described in yeast, in comparison of the unique PrP protein in higher eukaryotes. The presence of the PrP prion is associated with mammalian diseases. Whether fungal prions are...

présentation orale par N. Westbrook

[This corrects the article on p. e19500 in vol. 6.].

List of the oligonucleotides used in the study. Restriction sites are underlined. The CAA mutation of the IMP3 stop codon is in bold. (DOC)

Relative abundance of the 40S and 60S subunits. Determination of the ribosomal subunit ratio in the wild-type FS1 strain, the mutant IMPQ strain, and the complemented IMPQ strain carrying the pCEN-IMP3 vector. (TIF)

Drug sensitivity of the IMPQ strain. Growth of the FS1 and IMPQ strains transformed by an empty pCEN-URA3 vector (pFL38 vector), as well as the complemented IMPQ strain carrying the pCEN-IMP3 vector, is shown on arginine 0.2% minimum medium plates without uracil, and containing various concentrations of DNA damaging agents (Phleomycin 1 µg/mL, Bleo...

Analysis of telomere length. (A) The 1 kb DNA ladder is shown on the right panel. The upper signal on the left panel is an internal size reference. Lower bands are XhoI-digested telomeric DNA fragments revealed with specific telomeric probe. At least two independent DNA extractions are shown for each strain. est3Δ corresponds to strains deleted for...

Levels of the U3 snoRNA in the FS1 and IMPQ strains, and interaction between the Imp3 and Mpp10 proteins. (A) Northern blot analysis of the U3 snoRNA. Positions of the U3 and SCR1 RNA are indicated, as well as the U3/SCR1 ratio in percent. (B) Two-hybrid analysis. The growth of diploid strains carrying the indicated vectors is presented on minimal...

(DOC)

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 s...

Translation termination is usually a very efficient process. When a stop codon enters the ribosomal A-site it is recognized by the termination complex which promotes release of the polypeptide and dissociation of the ribosome. However, the efficiency of termination depends of the local context of the stop codon. In a number of cases, programmed sto...

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. Translational errors at a stop codon may also be i...

Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from P site to exit site is catalyzed in eukaryotes by the translocase elongation factor 2 (EF-2) and in prokaryotes by its homolog EF-G. During tRNA moveme...

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, coupli...