The cap-proximal RNA secondary structure inhibits preinitiation complex formation on HAC1 mRNA.

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.

Detection of HAC1 mRNA Splicing by RT-PCR in Saccharomyces cerevisiae.

HAC1 mRNA remains translationally repressed in the cytoplasm of the budding yeast Saccharomyces cerevisiae. Under conditions of cellular stress, a dual kinase RNase IRE1 (Inositol Requiring Enzyme-1) cleaves out an intervening sequence from the HAC1 mRNA. Cleaved mRNAs are then ligated by tRNA ligase, thus generating a spliced mRNA that translates an active transcription factor. This unconventional splicing of HAC1 mRNA in the cytoplasm is a molecular marker for various cellular stresses including oxidative stress and endoplasmic reticulum (ER) stress. This article describes a PCR-based protocol to detect the HAC1 mRNA splicing.

Phosphorylation of Pal2 by the protein kinases Kin1 and Kin2 modulates HAC1 mRNA splicing in the unfolded protein response in yeast.

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 nonphosphorylatable 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.

Vps34 and TOR Kinases Coordinate HAC1 mRNA Translation in the Presence or Absence of Ire1-Dependent Splicing.

In the budding yeast Saccharomyces cerevisiae, an mRNA, called HAC1, exists in a translationally repressed form in the cytoplasm. Under conditions of cellular stress, such as when unfolded proteins accumulate inside the endoplasmic reticulum (ER), an RNase Ire1 removes an intervening sequence (intron) from the HAC1 mRNA by nonconventional cytosolic splicing. Removal of the intron results in translational derepression of HAC1 mRNA and production of a transcription factor that activates expression of many enzymes and chaperones to increase the protein-folding capacity of the cell. Here, we show that Ire1-mediated RNA cleavage requires Watson-Crick base pairs in two RNA hairpins, which are located at the HAC1 mRNA exon-intron junctions. Then, we show that the translational derepression of HAC1 mRNA can occur independent of cytosolic splicing. These results are obtained from HAC1 variants that translated an active Hac1 protein from the unspliced mRNA. Additionally, we show that the phosphatidylinositol-3-kinase Vps34 and the nutrient-sensing kinases TOR and GCN2 are key regulators of HAC1 mRNA translation and consequently the ER stress responses. Collectively, our data suggest that the cytosolic splicing and the translational derepression of HAC1 mRNA are coordinated by unique and parallel networks of signaling pathways.

Phosphorylation of translation initiation factor eIF2α at Ser51 depends on site- and context-specific information.

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.

Chemical chaperone, TUDCA unlike PBA, mitigates protein aggregation efficiently and resists ER and non-ER stress induced HepG2 cell death.

Stress induced BSA (bovine serum albumin) protein aggregation is effectively mitigated in vitro by TUDCA (tauroursodeoxycholic acid) than by PBA (4- phenylbutyric acid), chemical chaperones approved by FDA for the treatment of biliary cirrhosis and urea cycle disorders respectively. TUDCA, unlike PBA, enhances trypsin mediated digestion of BSA. TUDCA activates PERK, an ER-resident kinase that phosphorylates the alpha-subunit of eukaryotic initiation factor2 (eIF2α) and promotes the expression of activated transcription factor 4 (ATF4) in HepG2 cells. In contrast, PBA induced eIF2α phosphorylation is not mediated by PERK activation and results in low ATF4 expression. Neither chaperones promote expression of BiP, an ER chaperone, and CHOP (C/EBP homologous protein), downstream target of eIF2α-ATF4 pathway. Both chaperones mitigate tunicamycin induced PERK-eIF2α-ATF4-CHOP arm of UPR and expression of BiP. TUDCA, unlike PBA does not decrease cell viability and it also mitigates tunicamycin, UV-irradiation and PBA induced PARP (poly ADP-ribose polymerase) cleavage and cell death. These findings therefore suggest that TUDCA's antiapoptotic activity to protect HepG2 cells and PBA's activity that limits tumor cell progression may be important while considering their therapeutic potential.

Tauroursodeoxycholic acid prevents stress induced aggregation of proteins in vitro and promotes PERK activation in HepG2 cells.

Tauroursodeoxycholic acid (TUDCA) a bile salt and chemical chaperone reduces stress-induced aggregation of proteins; activates PERK [PKR (RNA-dependent protein kinase)-like ER (endoplasmic reticulum) kinase] or EIF2AK3, one of the hall marks of ER stress induced unfolded protein response (UPR) in human hepatoblastoma HepG2 cells; prevents heat and dithiothreitol (DTT) induced aggregation of BSA (bovine serum albumin), and reduces ANS (1-anilino-naphthalene-8-sulfonate) bound BSA fluorescence in vitro. TUDCA inactivates heat treated, but not the native EcoR1 enzyme, and reduces heat-induced aggregation and activity of COX-1 (cyclooxygenase enzyme-1) in vitro. These findings suggest that TUDCA binds to the hydrophobic regions of proteins and prevents their subsequent aggregation. This may stabilize unfolded proteins that can mount UPR or facilitate their degradation through cellular degradation pathways.

Interaction of cytochrome c with tRNA and other polynucleotides.

Interaction between tRNA and other polynucleotides with cytochrome c was studied by visible spectroscopy, fluorescence spectroscopy and gel mobility shift assay in view of the recently reported important regulatory role of tRNA in cytochrome c mediated apoptotic pathway. Visible spectroscopy showed perturbation in the heme binding environment in cytochrome c with tRNA binding. Fluorescence titrations indicated that cytochrome c binds to different polynucleotides with differing affinities. A weak binding was observed with single stranded polyribonucleotides and polydeoxyribonuleotides and strong binding with tRNA and double stranded DNA as indicated by extent of fluorescence quenching and binding constants. Calculation of thermodynamic binding parameters from fluorescence titrations indicated that three molecules of cytochrome c bound with one tRNA molecule with binding constant of 1.9 × 10(6) M(-1). The perturbation of cytochrome c structure caused by the binding of tRNA could be affecting its role in mediating apoptosis.