Olivier Namy

Evidence for rRNA 2′-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes

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, which proposes that cells produce ribosomes of different composition and displaying different translational properties. This work reveals that ribosomal RNA 2′-O-methylation can be modulated in human ribosomes, including at key functional sites for translation, and that changes in the 2′-O-methylation pattern control the intrinsic capabilities of ribosomes to translate mRNAs. This work directly demonstrates the existence of composition-modified ribosomes and their associated change in translational activity as conceptualized by the specialized ribosome concept. Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2′-O-methylation (2′-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2′-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2′-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2′-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2′-O-Me, we identified a repertoire of 2′-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2′-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2′-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2′-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells

ABSTRACT Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor 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 diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.

Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons

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 of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patients genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function. This study identified readthrough-recoded CFTR channels, the activity of which could be enhanced using CFTR modulators http://ow.ly/f7Gd30hBCeG

Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single molecule fluorescence microscopy

Protein synthesis is a complex multistep 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. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.

Deciphering the reading of the genetic code by near-cognate tRNA

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/U-rich codons. In this latter case, their roles appear to stabilize the codon–anticodon interaction, allowing them to reach an optimal energetic value close to that of other interacting tRNAs involving G/C-rich anticodons. In this work we demonstrate that, while helping an efficient translation of A/U-rich codons, modified nucleotides also allow certain unconventional base pairing to occur, as evidenced in the case of stop codon suppression. Some codons of the genetic code can be read not only by cognate, but also by near-cognate tRNAs. This flexibility is thought to be conferred mainly by a mismatch between the third base of the codon and the first of the anticodon (the so-called “wobble” position). However, this simplistic explanation underestimates the importance of nucleotide modifications in the decoding process. Using a system in which only near-cognate tRNAs can decode a specific codon, we investigated the role of six modifications of the anticodon, or adjacent nucleotides, of the tRNAs specific for Tyr, Gln, Lys, Trp, Cys, and Arg in Saccharomyces cerevisiae. Modifications almost systematically rendered these tRNAs able to act as near-cognate tRNAs at stop codons, even though they involve noncanonical base pairs, without markedly affecting their ability to decode cognate or near-cognate sense codons. These findings reveal an important effect of modifications to tRNA decoding with implications for understanding the flexibility of the genetic code.