Deciphering the mRNP at premature termination codons

该项目的总体目标是全面破译导致NMD最终导致mRNA降解的过早翻译终止的分子事件。我们将建立在我们在第一个资助期建立或开发的试验系统的基础上。该系统包括完全重组的体外翻译系统、选择性核糖体图谱、NMD因子结合核糖体的亲和纯化和质谱仪(AP-MS)，以及结构研究。作为特定的目的，我们将首先更深入地阐明UPF3B在翻译终止中的作用条件，并直接探讨SMG6作为一种依赖于翻译终止的mRNA内切酶的即时功能和相互作用网络。其次，我们将使用基因组编辑的细胞系来亲和纯化释放因子和NMD因子结合的翻译核糖体，随后(1)通过质谱仪(AP-MS)和(2)核糖体图谱，从而有助于更深入地了解组织和转录本特异性的NMD分支，并有助于发现未知的蛋白质：蛋白质和蛋白质：在过早终止的核糖体上的蛋白质：mRNA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.