Nonsense-mediated mRNA decay (NMD) is an important quality control pathway conserved throughout all eukaryotes: mRNAs which contain premature stop codons (PTC) are detected and committed to accelerated decay to protect the cell from deleterious effects of truncated proteins. NMD also controls abundance of many wild-type genes that contain features recognized by the NMD machinery. Due to the important tasks of NMD, its proper functioning is essential for embryogenesis in vertebrates. Furthermore, NMD plays a prominent role in carcinogenesis and is involved in several genetic disorders.
The core NMD factors are identified and a picture of the steps involved is emerging. However, the exact interactions of the NMD factors, the order and orchestration of the processes, and the connection between PTC-recognition and mRNA decay remain unclear. In fact, detailed knowledge about NMD is necessary for drug development and more generally might reveal aspects of control and interactions in complex pathways that could be transferred to other systems.
I will dissect the interactions between factors that come into play after PTC-recognition, namely Smg5, Smg6, Smg7, Upf1 and the phosphatase PP2A. First, I will define their interacting domains by co-immunoprecipitation from human cells. Subsequently, I will reconstitute the complexes in vitro, determine binding affinities and try to solve their crystal structure. Mutations that disrupt these complexes will be used to test their role in NMD in vivo. The effect of Upf1-phosphorylation on these interactions will also be investigated. Furthermore, I plan to identify factors that connect PTC-recognition to the general mRNA degradation machineries by a co-immunoprecipitation screen from human cells.
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