New tools to isolate from cell extracts functional intermediates of the translation initiation process suitable for cryo-EM studies.
Angelita Simonetti1, Jailson Brito Querido1, Eder Mancera-Martínez1, Laurianne Kuhn2, Vicens Quentin and Yaser Hashem1
1 CNRS, Architecture et Réactivité de l’ARN UPR9002, Université de Strasbourg, 67084 Strasbourg, France.
2Platforme Protéomique Strasbourg-Esplanade, Centre National de la Recherche Scientifique, FRC 1589, Université de Strasbourg, Strasbourg, France
Translation initiation is a sophisticated process leading to ribosome assembly on the start codon. In Eukaryotes, the multiple pathways for start codon recognition require characteristic plethora of initiation factors (eIFs), which are selected depending on mRNA features, to guide the chronologically ordered assembly of interconverting ribosomal complexes. Up to now, the most commonly used strategy adopted for structural studies, has been the use of in vitro reconstituted complexes with a limited set of purified factors, but new factors specific for different mRNA signals are continuously discovered.
In the last years we have developed two strategies to investigate the structures and the molecular composition of translation intermediate complexes by cryo-EM and mass spectrometry analysis without any a priori prejudice on the eIFs composition. The strength of both strategies relies on the capability to block and isolate 48S and 80SICs bound on natural mRNAs (i.e. globine, H4 mRNA and IRES-containing viral and cellular mRNAs) directly from crude cell extracts thus leading to the identification and structural characterization of specific set of eIFs or new factors, which could even be tissue-specific (as some ITAFs).
Both techniques have been tested on different translation initiation mechanisms, revealing new functions for known initiation factors, new players and previously overlooked ribosomal features.