Deciphering the mRNP at premature termination codons

该项目的总体目标是全面破译最终导致NMD降解mRNA的过早翻译终止的分子事件。我们将以我们在第一个供资期内建立或开发的实验系统为基础。该系统包括一个完全重建的体外翻译系统，选择性核糖体分析，亲和纯化NMD因子结合的核糖体，然后进行质谱分析（AP-MS），和结构研究。作为具体的目标，我们将首先阐明UPF 3B在翻译终止中的作用条件，并将直接探讨SMG 6作为终止依赖性mRNA内切酶的直接功能以及相互作用网络。其次，我们将采用基因组编辑的细胞系亲和纯化释放因子和NMD因子结合的翻译核糖体，然后（1）进行质谱分析（AP-MS）和（2）进行核糖体分析，从而有助于更深入地了解组织和转录物特异性NMD分支，并发现未知的蛋白质：蛋白质和蛋白质：mRNA在过早终止核糖体的相互作用。

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.