In eukaryotic cells, the untoward accumulation of misfolded proteins inside the endoplasmic reticulum (ER) triggers a transcriptional program that restores cellular homeostasis. This program, called the unfolded protein response (UPR), is activated through a series of enzymatic steps that culminate in the production of a transcription factor that upregulates a set of target genes required for cell survival during ER stress. UPR activation occurs through an ER-resident sensor, Ire1, which is stimulated by misfolded proteins in the ER lumen. Activated Ire1 transmits a signal to its cytosolic portion, containing a kinase and an RNase, which trans-autophosphorylates and activates the RNase to cleave an mRNA encoding the Hac1 transcription factor. These cleavage products are ligated by tRNA ligase, Trl1, to produce a spliced form of HAC1 mRNA that is translated into the transcription factor. Together, the coordinated activation of Ire1 and ligation by Trl1 to produce spliced HAC1 mRNA comprise the central mechanism of UPR activation. This thesis describes my work toward understanding two key regulatory steps in UPR activation.

Several lines of evidence suggest that Ire1 kinase plays a role in the UPR that is autonomous from its role in RNase activation. First, RNase L, a close homolog of Ire1, is an RNase with a pseudokinase domain that has lost activity through evolution. Conservation of Ire1's kinase suggests that its activity is important for UPR fitness. Moreover, in vitro studies have shown that kinase inactive Ire1 retains RNase activity and, in vivo, kinase activity can be bypassed by ATP-competitive drugs. In an endeavor to understand the importance of Ire1 kinase we discovered that kinase activity contributes to the attenuation of Ire1 signaling and is imperative for homeostatic adaptation during ER stress.

The final chapters of this thesis describe my work to decipher how tRNA ligase achieves pathway specificity for the UPR. Trl1 splits its time between HAC1 mRNA splicing and tRNA splicing. Our efforts to discern how Trl1 acts in HAC1 mRNA splicing were unfruitful, however, this work enlightened our knowledge of Trl1 biology and set the stage for future work to elucidate Trl1's role in the UPR.