A Study on the Cellular Localization of Factors Involved in Yeast Nonsense-Mediated mRNA Decay and their Mechanisms of Control on Nonsense mRNA Translation: a Dissertation

Nonsense-mediated mRA decay (NM) is an importt mRA surveilance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NM pathway. One importnt aspect of NM which is presently the subject of intense controversy is the subcellular localization ofNM. In one set of experients, the decay kinetics of the ade2and pgkl nonsense mRAs (substrates for NM) were investigated in response to activating the NM pathway to determine if cytoplasmic nonsense mRAs are immune to NM in the yeast system. The results of these studies demonstrated that activation ofNM caused rapid and imediate degradation of both the ade2and the early nonsense pgkl steady state mRA populations. The half lives of the steady state mRA populations for both ade2and pgkl (early nonsense) were shortened from:; 30 minutes to approxiately 7 minutes. This was not observed for pgkl mRAs that contained a late nonsense codon demonstrating that activation of specifically targeted the proper substrates in these experiments. Therefore, in yeast nonsense mRAs residing in the cytoplasm are susceptible to NM. While these findings are consistent with NM occurng in the cytoplasm, they do not completely rule out the possibility of a nuclear-associated decay mechansm. To investigate the involvement of the nucleus in NM, the putative nuclear targeting sequence identified in Nmd2p (one of the trans-acting factors essential for NM) was characterized. Subcellular fractionation experients demonstrated that the jority of Nmd2p localized to the cytoplasm with a small proportion detected in the nucleus. Specific mutations in the putative nuclear localization signal (NS) of Nmd2p were found to have adverse effects on the protein s decay function. These effects on decay function, however, could not be attbuted to a failure in nuclear localization. Therefore , the residues that comprise the putative NLS of Nmd2p are important for decay fuction but do not appear to be required for tageting the protein to the nucleus. These results are in accordance with the findings above which implicate the cytoplasm as an important cellular comparent for NM. This thesis then investigates the regulatory roles of the trans-acting factors involved in NM (Upfl p, Nmd2p, and Upf3p) using a novel quatitative assay for translational suppression, based on a nonsense allele of the CANI gene (canlIOO). Deletion of UPFI, N1v2 or UPF3 stabilized the canlIOO trscript and promoted canJIOO nonsense suppression. Changes in mRA levels were not the basis of suppression, however, since deletion of DCPI or XRI or high-copy canlJOO expression in wild-ty cells caused mRA stabilization similar to that obtained in upjlnmd cells but did not result in comparable suppression. canlIOO suppression was highest in cells harboring a deletion of UP F 1 and overexpression of UP F 1 in cells with individual or multiple upjlnmd mutations lowered the level of nonsense suppression without affecting the abundace of the canlIOO mRA. These findings indicate that Nmd2p and Upf3p reguate Upfl p activity and that Upfl p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRA decay.