Genetic dissection of nonsense-mediated MRNA decay in drosophila

The nonsense-mediated mRNA decay (NMD) pathway functions as a quality control mechanism and a feature of post-transcriptional gene regulation. NMD degrades mRNAs containing premature termination codons (PTCs) to prevent the production of potentially harmful truncated proteins, and it also destroys many error-free endogenous mRNAs to limit the expression of these genes. NMD is critical for viability in most complex organisms, highlighting the importance of this pathway; however, it is unknown which of the two NMD functions is the feature essential for viability. Understanding how NMD recognizes and degrades targets may provide insight to uncover the requirement of this pathway for viability, but the molecular mechanisms of NMD target recognition and destruction also remain unclear. Work presented in this dissertation describes genetic analysis in Drosophila to reveal the feature of the NMD pathway that is critical for viability, and refines the model describing the mechanism of NMD target degradation. We screened a collection of heterozygous deficiencies for suppression of the incomplete lethality of a hypomorphic allele of the core NMD factor Upf2. This screen identified three autosomal regions that partially suppress Upf2 mutant lethality when deleted. The endogenous NMD target Gadd45 is located within one suppressing region, and we found that elimination of Gadd45 restores viability to multiple null NMD mutants. Mekk1, a factor that acts downstream of Gadd45, resides in another suppressing region, and loss of Mekk1 also restores viability to NMD mutants. The third suppressing region contains ! ! iv! Arc2, and we found that Arc2 and the closely related Arc1 may also contribute to the lethality of Drosophila lacking NMD activity. In addition, this dissertation describes the first genetic analysis of Drosophila Smg5 mutants, and determines that Smg5 is a critical NMD factor required for viability and all NMD function. Further analysis of the first characterization of double mutants for multiple NMD factors reveals there are multiple mechanisms for NMD target degradation. These findings provide a new foundation for understanding the crucial NMD gene regulatory function and reshape the model of the NMD pathway.