Modulation of translation initiation by eIF4A

Protein synthesis, or translation, is regulated at the initiation level by the recruitment of the 40S ribosomal subunit and associated factors to the 5’ end of the mRNAs. This process is catalyzed by the eukaryotic Initiation Factor 4F (eIF4F) complex (composed by the cap-binding protein, eIF4E; a scaffolding protein, eIF4G; and the RNA helicase, eIF4A). eIF4A is thought to unwind 5’UTRs secondary structure from the mRNAs, facilitating ribosome binding. This helicase has two isoforms that are considered equivalent according to evidence from in vitro assays. Here, we took advantage of RNAi and pharmacological approaches to demonstrate that the eIF4A isoforms are not equivalent in vivo. We found that eIF4AI, but not eIF4AII, suppression leads to inhibition of proliferation in Eu-Myc lymphoma cells and that the increase in eIF4AII protein levels cannot compensate for the absence of eIF4AI. Furthermore it was demonstrated that eIF4AII transcription is linked to eIF4AI expression.Later, we examined the performance of eIF4A isoforms in a cell differentiation system. We documented that eIF4AII, but not eIF4AI, levels increase during muscle cell differentiation. As well, only eIF4AII isoform is under MyoD (master regulator of muscle differentiation). Furthermore it was determined that eIF4A activity is necessary for muscle cell differentiation process to occur.Silvestrol is a small molecule that has exhibited chemotherapeutic potential. This compound inhibits translation by targeting the helicase eIF4A; however controversies about its mechanism of action have arisen. Here, via gene-wide screening experiments in yeast, it was demonstrated that TIF1/2 (yeast eIF4A) is the main target of rocaglamide compounds. Furthermore, a model in which the binding site of silvestrol is near eIF4A RNA binding site is proposed.La synthèse protéique, ou la traduction, est régulé au niveau de l'initiation par le recrutement du ribosome à l’ARNm. Cette phase est catalysée par le complexe de facteurs d'initiation 4F (eIF4F) (composé de trois sous unités: eIF4E, eIF4G, et eIF4A).La protéine helicase eIF4A défait les structures secondaires de l’ARNm pour faciliter l’attachement de la sous unité ribosomique 40S. eIF4A a deux isoformes qui sont considérés équivalent, soutenue par plusieurs expériences menées in vitro. Ici, nous avons profité de l'ARNi et des approches pharmacologiques pour démontrer que ces isoformes ne sont pas équivalentes in vivo. Nous avons constaté que la suppression de eIF4AI, mais pas eIF4AII, conduit à l'inhibition de la prolifération des cellules de lymphome. Aussi, nous avons observé que l'augmentation des taux de protéine eIF4AII ne peut pas compenser pour les fonctions du eIF4A quand il n’est pas présent dans la cellule. En outre, il a été démontré que la transcription de eIF4AII est liée à l'expression de eIF4AI.Plus tard, nous avons examiné la performance des isoformes eIF4A dans un système de différenciation cellulaire. Nous avons documenté l’augmentation des niveaux de protéine eIF4AII pendant la différentiation des cellules musculaires, et que seuls eIF4AII est sous contrôle de MyoD au cours du processus. De plus, il a été déterminé que l'activité d’eIF4A est nécessaire pour que la différenciation des cellules musculaires se produise.Silvestrol est une petite molécule qui a exposé potentiel chimio thérapeutique. Cette molecule inhibe la traduction en ciblant eIF4A; mais des controverses concernant son mécanisme d'action ont surgi récemment. En utilisant deux approches de dépistage génétiques, il a été démontré que TIF1/2 (S. cerevisiae eIF4A) est la cible principale de silvestrol. Finalement, un modèle dans lequel le site de liaison silvestrol est à proximité du site de liaison d'ARN est proposé

Evidence for a functionally relevant rocaglamide binding site on the eIF4A:RNA complex

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway, however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context, and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides, and defined a discrete set of residues near the RNA binding motif which confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease

Evidence for a Functionally Relevant Rocaglamide Binding Site on the eIF4A–RNA Complex

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and <i>in silico</i> modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, <i>in silico</i> modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease