The Importance of tRNA Modification and SSD1 Status for Protein Homeostasis and Cell Viability

Chemical modifications of anticodon loop are required to support tRNA function in mRNA decoding and to prevent protein aggregation in the eukaryotic model system Saccharomyces cerevisiae. This work investigates the effects of mutation in the gene encoding the Deg1 pseudouridine synthase. It is known that the mutation of the human DEG1 orthologue PUS3 causes severe neurological and developmental defects, emphasizing the general importance of the modification. However, the exact cellular consequences of deg1/pus3mutation remain to be resolved. This thesis shows that in yeast, the absence of a functional Deg1 synthase induces slow growth, heat and drug sensitivities and protein homeostasis defects. In addition, depending on the specific selection of the yeast strain background, a shortened chronological lifespan could also be observed, and strong negative genetic interactions of DEG1with other genes involved in anticodon loop modification were detected. Strain background influence on phenotypic variation is partly due to allelic variation of the gene encoding the RNA binding protein Ssd1. A common laboratory yeast strain used in this work contains the ssd1-d allele encoding a truncated non- functional Ssd1 variant. This allele is linked to phenotypes that partially overlap with those of deg1. Thus, enhancing deg1 phenotypes by ssd1-dmay result from the additivity of negative phenotypes and may not necessarily involve shared molecular mechanisms. Since protein homeostasis defects could be functionally involved in aging phenotypes, life spans and protein aggregation in deg1 mutants and strains lacking a ribosome-associated chaperone involved in co-translational protein folding is compared. While both strains accumulated protein aggregates, only the deg1 mutant exhibited a reduced life span. Hence, other cellular consequences than protein aggregation may cause the deg1 aging phenotype. Since yeast’s lifespan is also known to be affected by the TOR (target of rapamycin) kinase activity, the rapamycin sensitivities and autophagy (a process under the direct control of TOR) were analyzed. The absence of DEG1 is found to induce rapamycin sensitivity and increased autophagy. Both effects are enhanced in the absence of functional Ssd1, pointing to a variation of TOR activity in the different yeast strain backgrounds. Direct translational effects of the deg1mutation were studied using a glutamine-rich Rnq1 protein. The results suggest that the combined absence of functional SSD1 and DEG1 aggravates the expression defect of the protein, suggesting that translation is negatively affected by mutation in both genes.