Final Report Summary - COMPLEXNMD (NMD Complexes: Eukaryotic mRNA Quality Control) Nonsense-mediated mRNA decay (NMD) is an essential surveillance mechanism in the eukaryotic cell, eliminating faulty messenger RNA (mRNA) with a premature stop codon (PTC). Moreover, NMD is involved in regulation of gene expression of about 5-20% of normal mRNA transcripts. NMD is a highly complex process and different tissue- and transcript-specific NMD pathways were proposed. A wide range of genetic diseases have their origin in the mechanisms of NMD. Discrimination of a PTC from a correct termination codon is the key step in NMD. This key step is known to depend on active translation, but the molecular mechanisms and dynamics of this process remain poorly understood. We established a human cell-free translation system from purified factors which provides a defined system to study human translation termination and the impact of NMD factors. We investigated the specific interactions of PABPC1 and the eukaryotic release factors in the context of a terminating ribosome at a normal stop codon and showed a specific stimulation of stop codon recognition. Moreover, we studied the impact of the conserved NMD factors (UPF proteins) on termination. Unexpectedly, we found that the key NMD factor UPF1 does not interact with eukaryotic release factors as previously reported. It also does not interfere with translation termination directly or bind next to the ribosome close to the active site. This contradicted current NMD models and required substantial further investigations and control experiments in order to show that our purified UPF1 protein is active and that our findings are independent from the phosphorylation state of UPF1.In fact, UPF1 activation to trigger mRNA decay requires phosphorylation by the SMG-1-8-9 kinase complex. By cross linking / mass spectrometry and biochemical assays, we showed that the activity of the kinase SMG-1 is controlled by the regulatory subunits SMG-8 and SMG-9 which interact with an insertion domain that may have the role of a gatekeeper for substrates. We visualized the binding of UPF1 to the SMG-1-8-9 kinase complex by electron cryo-microscopy. Moreover, we identified additional levels of regulation controlling the activity of SMG-1 kinase by the other conserved NMD factors UPF2 and UPF3B. Using our in vitro reconstituted mammalian translation system, recombinant production of human multiprotein complexes by MultiBac insect cell expression, toe-printing and peptide release assays, we obtained new insights on how UPF1 and SMG-1-8-9 kinase may be recruited to the PTC-bound ribosome. We visualized important ribosomal complexes by electron cryo-microscopy providing important mechanistic insights. Taken together, our experiments suggest a mechanistic model of recognition of a PTC and activation of mRNA decay by the NMD machinery which builds on and modifies current NMD models providing an improved understanding at a molecular level of this paramount step in human translational control. This is a vital prerequisite for the development of new therapies and intervention strategies for treating NMD-related diseases.
