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Translation of HAC1 mRNA in the budding yeast Saccharomyces cerevisiae is derepressed when RNase Ire1 removes its intron via nonconventional cytosolic splicing in response to accumulation of unfolded proteins inside the endoplasmic reticulum. The spliced HAC1 mRNA is translated into a transcription factor that changes the cellular gene expression patterns to increase the protein folding capacity of cells. Previously, we showed that a segment of the intronic sequence interacts with the 5'-UTR of the unspliced mRNA, resulting in repression of HAC1 translation at the initiation stage. However, the exact mechanism of translational derepression is not clear. Here, we show that at least 11-base-pairing interactions between the 5'-UTR and intron (UI) are sufficient to repress HAC1 translation. We also show that overexpression of the helicase eukaryotic initiation factor 4A derepressed translation of an unspliced HAC1 mRNA containing only 11-bp interactions between the 5'-UTR and intronic sequences. In addition, our genetic screen identifies that single mutations in the UI interaction site could derepress translation of the unspliced HAC1 mRNA. Furthermore, we show that the addition of 24 RNA bases between the mRNA 5'-cap and the UI interaction site derepressed translation of the unspliced HAC1 mRNA. Together, our data provide a mechanistic explanation for why the cap-proximal UI-RNA duplex inhibits the recruitment of translating ribosomes to HAC1 mRNA, thus keeping mRNA translationally repressed.

During cellular stress in the budding yeast Saccharomyces cerevisiae, an endoplasmic reticulum (ER)-resident dual kinase and RNase Ire1 splices an intron from HAC1 mRNA in the cytosol, thereby releasing its translational block. Hac1 protein then activates an adaptive cellular stress response called the unfolded protein response (UPR) that maintains ER homeostasis. The polarity-inducing protein kinases Kin1 and Kin2 contribute to HAC1 mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediated HAC1 mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3’-untranslated region (3’-UTR) of HAC1 mRNA, and a yeast strain lacking both Pal1 and Pal2 was deficient in HAC1 mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a non-phosphorylatable Pal2 mutant could not rescue the UPR defect in a pal1Δ pal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinates HAC1 mRNA processing and ER homeostasis.

Protein kinases phosphorylate specific amino acid residues of substrate proteins and regulate many cellular processes. Specificity for phosphorylation depends on the accessibility of these residues, and more importantly, kinases have preferences for certain residues flanking the phospho-acceptor site. Translation initiation factor 2α [eukaryotic translation initiation factor 2α (eIF2α)] kinase phosphorylates serine51 (Ser51) of eIF2α and downregulates cellular protein synthesis. Structural information on eIF2α reveals that Ser51 is located within a flexible loop, referred to as the Ser51 loop. Recently, we have shown that conformational change of the Ser51 loop increases the accessibility of Ser51 to the kinase active site for phosphorylation. Here, we show that the specificity of Ser51 phosphorylation depends largely on its relative position in the Ser51 loop and minimally on the flanking residues.

Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed ER stress, activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog, Kin2, in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity-regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an autoinhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for UPR function. We also show that the functional mini-Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that transphosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for full kinase activity. Collectively, our data suggest that, during ER stress, the Kin kinase domain is released from its autoinhibitory KA1 domain and is activated by transphosphorylation.