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.

Early Changes in Immune Cell Count, Metabolism, and Function Following Sleeve Gastrectomy: A Prospective Human Study.

OBJECTIVE: To characterize longitudinal changes in blood biomarkers, leukocyte composition, and gene expression following laparoscopic sleeve gastrectomy (LSG). BACKGROUND: LSG is an effective treatment for obesity, leading to sustainable weight loss and improvements in obesity-related comorbidities and inflammatory profiles. However, the effects of LSG on immune function and metabolism remain uncertain. METHODS: Prospective data were collected from 23 enrolled human subjects from a single institution. Parameters of weight, comorbidities, and trends in blood biomarkers and leukocyte subsets were observed from preoperative baseline to 1 year postsurgery in 3-month follow-up intervals. RNA sequencing was performed on pairs of whole blood samples from the first 6 subjects of the study (baseline and 3 months postsurgery) to identify genome-wide gene expression changes associated with undergoing LSG. RESULTS: LSG led to a significant decrease in mean total body weight loss (18.1%) at 3 months and among diabetic subjects a reduction in hemoglobin A1c. Improvements in clinical inflammatory and hormonal biomarkers were demonstrated as early as 3 months after LSG. A reduction in neutrophil-lymphocyte ratio was observed, driven by a reduction in absolute neutrophil counts. Gene set enrichment analyses of differential whole blood gene expression demonstrated that after 3 months LSG induced transcriptomic changes not only in inflammatory cytokine pathways but also in several key metabolic pathways related to energy metabolism. CONCLUSIONS: LSG induces significant changes in the composition and metabolism of immune cells as early as 3 months postoperatively. Further evaluation is required of bariatric surgery's effects on immunometabolism and the consequences for host defense and metabolic disease.

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.

Early changes in immune cell metabolism and function are a hallmark of sleeve gastrectomy: a prospective human study.

OBJECTIVE: To characterize longitudinal changes in blood biomarkers, leukocyte composition, and gene expression following laparoscopic sleeve gastrectomy (LSG). BACKGROUND: LSG is an effective treatment for obesity, leading to sustainable weight loss and improvements in obesity-related co-morbidities and inflammatory profiles. However, the effects of LSG on immune function and metabolism remain uncertain. METHODS: Prospective data was collected from 23 enrolled human subjects from a single institution. Parameters of weight, co-morbidities, and trends in blood biomarkers and leukocyte subsets were observed from pre-operative baseline to one year in three-month follow-up intervals. RNA-sequencing was performed on pairs of whole blood samples from the first six subjects of the study (baseline and three months post-surgery) to identify genome-wide gene expression changes associated with undergoing LSG. RESULTS: LSG led to a significant decrease in mean total body weight loss (18.1%) at three months and among diabetic subjects a reduction in HbA1c. Improvements in clinical inflammatory and hormonal biomarkers were demonstrated as early as three months after LSG. A reduction in neutrophil-lymphocyte ratio was observed, driven by a reduction in absolute neutrophil counts. Gene set enrichment analyses of differential whole blood gene expression demonstrated that after three months, LSG induced transcriptomic changes not only in inflammatory cytokine pathways but also in several key metabolic pathways related to energy metabolism. CONCLUSIONS: LSG induces significant changes in the composition and metabolism of immune cells as early as three months post-operatively. Further evaluation is required of bariatric surgery's effects on immunometabolism and consequences for host defense and metabolic disease.

Adaptation to Endoplasmic Reticulum Stress Requires Transphosphorylation within the Activation Loop of Protein Kinases Kin1 and Kin2, Orthologs of Human Microtubule Affinity-Regulating Kinase.

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.

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.

Evidence That Base-pairing Interaction between Intron and mRNA Leader Sequences Inhibits Initiation of HAC1 mRNA Translation in Yeast.

The Hac1 transcription factor in yeast up-regulates a collection of genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the 5'-untranslated region (5' UTR) of the HAC1 mRNA represses Hac1 protein production under basal conditions, whereas cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under endoplasmic reticulum stress conditions, relieves the inhibition and enables Hac1 synthesis. Using a random mutational screen as well as site-directed mutagenesis, we identify point mutations within the 5' UTR-intron interaction site that derepress translation of the unspliced HAC1 mRNA. We also show that insertion of an in-frame AUG start codon upstream of the interaction site releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. These results suggest that the major block of translation occurs at the initiation stage. Supporting this interpretation, the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes versus free ribosomal subunits. Thus, our results provide evidence that the 5' UTR-intron interaction represses translation initiation on the unspliced HAC1 mRNA.