Identification and characterisation of novel involved in the nonsense-mediated mRNA decay (NMD) pathway
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Nonsense mediated mRNA decay (NMD) is a surveillance mechanism that targets transcripts containing premature stop codons (PTCs) for degradation, and that also regulates up to 10% of the whole transcriptome. During the course of my PhD I set out to identify novel NMD factors by performing a genome-wide RNA interference (RNAi) screen in a transgenic strain of Caenorhabditis elegans carrying an NMD reporter. I identified five novel proteins that are putative NMD factors in worms: NGP-1, NPP-20, AEX-6, PBS-2 and NOAH-2. Knock-down of these proteins led to severe developmental defects: worms were either arrested during various larval stages or died prematurely. The only exception was AEX-6, the knockdown of which led to a milder phenotype. Homology analysis of the novel C. elegans NMD factors showed that these proteins are conserved in human, with the exception of NOAH-2, which only has a homologue in Drosophila melanogaster, NOMPA. By performing an NMD assay in human cells, I demonstrated that GNL2 (NGP-1) and SEC13 (NPP-20) are functionally conserved NMD factors in human. Analysis of the consequences of depletion of GNL2, SEC13, UPF1 or UPF2 on the transcriptome of HeLa cells revealed that these four proteins co-regulate a subset of endogenous NMD targets, whilst also independently regulating the expression of other sets of transcripts. The findings presented in this thesis further our knowledge of the biology of NMD in both nematodes and humans. They demonstrate the existence of further regulators of this surveillance pathway, and add a layer of complexity to this fine-tuned biological process.