UCSF

In eukaryotic cells, all secreted and transmembrane proteins are trafficked through the endoplasmic reticulum (ER). They are accurately translated, folded and modified within the specialized environment of the ER. The fidelity of protein processing within the ER is crucial for cell survival. Therefore, mechanisms have evolved that regulate translation, translocation and proper processing of newly synthesized proteins. The unfolded protein response (UPR) is one such pathway that monitors the protein folding capacity of the ER. In all organisms analyzed to date, the UPR drives transcriptional programs that allow cells to cope with ER stress. In budding yeast, the key regulatory step in this pathway is the non non-conventional splicing of HAC1 encoding a transcriptional activator. This splicing event is conserved in higher organisms and dependent on Ire1, an ER membrane-resident kinase/endoribonuclease. This thesis adds to the continuing story of the UPR by extending the study of this conserved pathway into the fission yeast Schizosaccharomyces pombe. We found that the fission yeast S. pombe lacks both a Hac1 ortholog and a UPR-dependent transcriptional program. Instead, Ire1 initiates the selective decay of a subset of ER-localized mRNAs that is required to survive ER stress. We identified Bip1 mRNA, encoding a major ER chaperone, as the sole mRNA cleaved upon Ire1 activation that escapes decay. Instead, truncation of its 3' UTR, including loss of its polyA tail, stabilized Bip1 mRNA, resulting in increased BiP translation. Thus, S. pombe uses a universally conserved stress-sensing machinery in novel ways to maintain homeostasis in the ER. Additionally, we present data that shows that mutations in the RNA degradation pathway in budding yeast are able to suppress the UPR specific growth defect of a mutant allele of tRNA ligase, trl1-100. We also studied the regulation of translation by genome wide high throughput ribosome footprinting in cells with harboring mutated ribosomes and cells lacking a key component of the machinery that targets proteins for co-translational translocation into the ER. Together the work presented here attempts to understand the role that mRNA regulation plays in various mechanisms regulating protein synthesis, translocation and ER homeostasis.