Colaboration with Prof. Dr. Kulozik and Prof. Dr. Hentze
PROJECT INFORMATION :
Nonsense-mediated mRNA decay (NMD) represents one of the core quality control mechanisms of gene expression, which eliminates faulty mRNAs encoding C-terminally truncated proteins. Extending beyond the basic biologic significance, NMD is also medically highly relevant because it represents an important modifier of inherited an aquired diseases. Therefore, it is important to decipher the molecular mechanisms that link NMD with translation termination. The goal of this project is to use our recently established cell-free translation system consisting of a short synthetic open reading frame and purified components of the translation termination apparatus and NMD proteins to gain critical insight into the molecular interactions that connect translation termination with the NMD machinery. As specific aims we will firstly directly address the question of how NMD factors affect the efficiency and fidelity of translation termination. Secondly, we will employ a two-step affinity purification scheme followed by mass spectrometry (AP-MS), to characterize the NMD factor - termination factor interactome at a terminating ribosome. Finally, using the same affinity purification scheme and ribosomal profiling, we will identify the transcripts associated with NMD factor – bound terminating ribosomes in different cell types, thus contributing to a deeper understanding of tissue and transcript specific NMD branches and to the discovery of so far unknown and protein-protein and protein-mRNA interactions at prematurely terminating ribosomes.
KEY TECHNOLOGIES :
- RNA interactome capture
- RNA analysis technologies
- Fully reconstituted in vitro translation
Hauer C, Sieber J, Schwarzl T, Hollerer I, Curk T, Alleaume A-M, Hentze MW, Kulozik AE. Mammalian exon junction complexes show a distributional bias towards alternatively spliced mRNAs and against mRNAs coding for ribosomal proteins. Cell Reports accepted (2016)
Sieber J, Hauer C, Bhuvanagiri M, Leicht S, Krijgsveld J, Neu-Yilik G, Hentze MW, Kulozik AE. Proteomic analysis reveals branch-specific regulation oft he unfolded protein response by nonsense-mediated mRNA decay. Mol. Cell. Proteomics 15: 1584-97 (2016)
Deniaud A, Karuppasamy M, Bock T, Masiulis S, Huard K, Garzoni F, Kerschgens K, Hentze MW, Kulozik AE, Beck M, Neu-Yilik G, Schaffitzel C. A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation. Nucleic Acids Nucleic Acids Research 43: 7600-11 (2015)
Hauer C, Curk T, Anders S, Schwarzl T, Alleaume AM, Sieber J, Hollerer I, Bhuvanagiri M, Huber W, Hentze MW, Kulozik AE. Improved binding site assignment by high-resolution mapping of RNA-protein interactions using iCLIP. Nature Communications 6:7921. (2015).
Neu-Yilik G, Amthor B, Gehring N, Bahri S, Paidassi H, Hentze MW, Kulozik AE. Mechanism of escape from nonsense-mediated decay of human β-globin mRNAs with early nonsense mutations. RNA 17:843-54 (2011)