Mechanisms of eukaryotic translation and ribosome-associated mRNA surveillance and protein quality control

Suny Downstate Medical Center, Brooklyn NY

Investigators

Linked publications & trials

Abstract

The focus of research in my laboratory is investigation of fundamental canonical and non-canonical mechanisms of mammalian and viral translation, as well as of the highly conserved mRNA and protein quality control processes that are induced by ribosomal stalling and include mRNA No-Go decay (NGD) surveillance which targets mRNAs on which ribosomes are stalled by e.g. stable secondary structures, rare codons or polyA stretches, and the corresponding ribosome-associated quality control (RQC) pathway that degrades aberrant, potentially toxic nascent chain polypeptides arising from interrupted translation. Our development of reagents and protocols for in vitro reconstitution of the entire mammalian translation process from individual purified components and our recent success in reconstituting RQC puts us in a position to close critical gaps in understanding of the molecular mechanisms of several individual stages in these processes and to begin detailed characterization of the mechanism of mammalian NGD. In pursuing these goals, we will continue to use the in vitro reconstitution approach combined with state-of-art biochemical techniques and will integrate our expertise with the technical advances in cryo-electron microscopy, including time-resolved cryo-EM, developed in the laboratory of J. Frank (Columbia University). In the canonical translation process, we will (i) investigate further the mechanism of action of DHX29 during ribosomal scanning on mRNAs with highly structured 5'UTRs as well as the suggested activities of DHX9 and DHX36 in initiation on mRNAs containing G-quadruplet structures, (ii) determine structural aspects of ABCE1-mediated ribosome recycling by time-resolved cryo-EM in collaboration with J. Frank, and (iii) investigate the mechanism of splitting of stored 80S ribosomes bound to hibernation factors. Concerning viral translation, we will focus on (i) analysis of the molecular mechanism of initiation on Type 1 IRESs that occur in important human pathogens e.g. rhinovirus and poliovirus which has oncolytic activity and is in trials for cancer therapy, and (ii) in vitro reconstitution and determination of the mechanism of initiation on SARS-CoV2 mRNA and elucidation of the mechanism by which this mRNA evades Nsp1-mediated endonucleolytic cleavage and translation inhibition. Further studies of RQC will include (i) determination of the mechanism by which TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains arising from interrupted translation to target them for degradation, (ii) recapitulation in vitro of nascent chain CAT tailing and investigation of the mechanism of this process, and (iii) characterization of the mechanism underlying the K63-specificity of ribosomal ubiquitination by ZNF598 and determination of its ribosomal position. Importantly, the ability to reconstitute RQC will now enable us to assay the activity of N4BP2, which has been suggested to be the mammalian orthologue of the S. cerevisiae NGD mRNA endonuclease Cue2, and to follow mammalian RQC-dependent and RQC-independent NGD branches.