This proposal focuses on the mechanisms of mammalian protein synthesis and ribosome-associated mRNA surveillance (No-Go and non-stop decay (NGD and NSD, respectively)) and protein quality control pathways. Translation is a cyclical process, consisting of initiation, elongation, termination and ribosome recycling. While elongation is highly conserved, other stages differ significantly between kingdoms. Initiation is the most complicated and regulated stage of mammalian translation involving a complex interplay between multiple initiation factors (eIFs), whereas termination and recycling are intimately connected with cellular mRNA and protein quality control systems that are induced by aberrant stalling of elongation complexes by e.g. stable secondary structures, rare codons, damaged RNA bases (NGD), or the absence of a stop codon (NSD). Our development of in vitro reconstitution of the entire mammalian translation process and recent integration of our expertise with technical advances in cryo-electron microscopy made in the laboratory of J. Frank (HHMI, Columbia University) now gives us a unique opportunity to close several critical gaps in understanding of the mechanism of mammalian translation, to investigate physiologically important cases of translational regulation, and to extend our studies to associated mRNA and protein quality control pathways. We will (i) address the unresolved mechanistic aspects of the key stages in the canonical translation process (e.g. ribosomal recruitment of Met-tRNAiMet and the role in it of ABC50, the mechanism of eIF4F-mediated attachment of 43S ribosomal preinitiation complexes to capped mRNAs, the mechanism of action of DHX29 during ribosomal scanning and kinetics of this process, and kinetics of mammalian termination), (ii) recapitulate in vitro and determine mechanisms of specific cases of non-canonical initiation that have high physiological importance or clinical relevance, such as initiation mediated by Leu-tRNALeu, repeat-associated non-AUG (RAN) translation, initiation on distinct cellular IRESs and regulation of 5?-terminal oligopyrimidine (TOP) mRNAs, and (iii) develop our recent advances concerning mechanisms of ribosome-associated mRNA and protein quality control, including the role of peptidyl-tRNA hydrolase PTRH1 in release of ribosome-associated nascent chain peptidyl- tRNAs arising from interrupted translation, and the mechanism of function of GTPBP1 and GTPBP2, members of a relatively divergent group of translational GTPases. We will continue to use the in vitro reconstitution approach, integrated with state-of-art biochemical techniques, as well as kinetic and structural methods in collaboration with J. Frank (HHMI, Columbia University), Y. Hashem (IBMC Strasbourg, France), and M. Rodnina (MPI Gttingen, Germany).
Protein synthesis is tightly regulated to ensure its accuracy and ability to accommodate dynamic changes in cellular metabolic demands. Defects in the translation apparatus and its regulation cause a variety of devastating diseases, including cancer and neurodegeneration, and translation is therefore increasingly becoming a focus for chemotherapeutic intervention. Determination of the molecular mechanisms of important steps in the translation process is essential for development of therapeutic approaches to ameliorate such defects.
|Lin, Kai Ying; Nag, Nabanita; Pestova, Tatyana V et al. (2018) Human eIF5 and eIF1A Compete for Binding to eIF5B. Biochemistry 57:5910-5920|
|Zinoviev, Alexandra; Goyal, Akanksha; Jindal, Supriya et al. (2018) Functions of unconventional mammalian translational GTPases GTPBP1 and GTPBP2. Genes Dev 32:1226-1241|