Deciphering the mRNP at premature termination codons

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The overall goal of this project is to comprehensively decipher the molecular events of premature translation termination that finally result in mRNA degradation by NMD. We will build on the experimental system that we have established or developed in the first funding period. This system includes a fully reconstituted in vitro translation system, selective ribosomal profiling, affinity purification of NMD factor-bound ribosomes followed by mass spectrometry (AP-MS), and structural studies. As specific aims, we will firstly elucidate the conditions of UPF3B function in translation termination in more depth and will directly probe the immediate function as well as the interaction network of SMG6 as a termination dependent mRNA endonuclease. Secondly, we will employ genome-edited cell lines to affinity-purify release factor- and NMD factor-bound translating ribosomes followed (1) by mass spectrometry (AP-MS) and (2) by ribosomal profiling thus contributing to a deeper understanding of tissue- and transcript-specific NMD branches and to the discovery of unknown protein:protein and protein:mRNA interactions at prematurely terminating ribosomes.