Discovery and analysis of components of nonsense mediated decay in drosophila

Nonsense mediated decay (NMD) is a cellular surveillance mechanism originally identified for its role in targeting mRNAs containing nonsense codons for rapid degradation. Nonsense codons, or premature termination codons (PTCs), are a common result of genomic mutation, alternative splicing, and programmed frameshifts that bring a premature termination codon into the reading frame. Degradation of the mRNA encoding PTCs is thought to prevent the translation and build-up of potentially harmful truncated proteins within the cell. The phenomenon of NMD is evolutionarily conserved, as destabilization of PTC-containing mRNA occurs in all eukaryotes examined. Transacting factors responsible for the destabilization of PTC-containing mRNA were identified in a series of informational suppressor screens performed in yeast and C. elegans. Combined, these screens identified seven genes that are conserved throughout eukaryotes. Homologs of genes identified in the original screens have been analyzed biochemically to determine how these trans-acting factors can recognize a premature termination codon. Two models of PTC-recognition have resulted from these biochemical studies; the primary model suggests a PTC causes aberrant translation termination that mimics an abnormally long 3'UTR. The second models suggests factors remain associated with the mRNA after intron splicing, and these factors recruit the NMD machinery to the mRNA, marking it for degradation. However, while analyzed biochemically, many questions remain about the mechanism of NMD. Have all the genes iv required for NMD been identified? What are the roles of NMD pathway genes in vivo? How does NMD differentiate a native termination codon from a premature termination codon? Work presented in this thesis focuses on these questions. Here we present a forward genetic screen in Drosophila, which isolated alleles of known and potentially novel components of the NMD pathway. We use alleles isolated from this screen to investigate the mechanism of NMD function in vivo. Finally, we analyze cis-acting sequences involved in NMD targeting in Drosophila.