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

   https://doi.org/10.1126/scisignal.aaz4401  

   Ghosh, Chandrima ; Uppala, Jagadeesh Kumar ; Sathe, Leena ; Hammond, Charlotte I. ; Anshu, Ashish ; Pokkuluri, P. Raj ; Turk, Benjamin E. ; Dey, Madhusudan ( May 2021 , Science Signaling)

   During cellular stress in the budding yeastSaccharomyces cerevisiae, an endoplasmic reticulum (ER)–resident dual kinase and RNase Ire1 splices an intron fromHAC1mRNA 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 toHAC1mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediatedHAC1mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3′ untranslated region (3′UTR) ofHAC1mRNA, and a yeast strain lacking both Pal1 and Pal2 was deficient inHAC1mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a nonphosphorylatable Pal2 mutant could not rescue the UPR defect in apal1Δpal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinatesHAC1mRNA processing and ER homeostasis.

    

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2. Specificity in endoplasmic reticulum-stress signaling in yeast entails a step-wise engagement of HAC1 mRNA to clusters of the stress sensor Ire1

   https://doi.org/10.7554/eLife.05031  

   van Anken, Eelco ; Pincus, David ; Coyle, Scott ; Aragón, Tomás ; Osman, Christof ; Lari, Federica ; Gómez Puerta, Silvia ; Korennykh, Alexei V ; Walter, Peter ( December 2014 , eLife)

   Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.

    

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3. Plant growth regulators interact with elevated temperature to alter heat stress signaling via the Unfolded Protein Response in maize

   https://doi.org/10.1038/s41598-019-46839-9  

   Neill, Elena M. ; Byrd, Michael C. R. ; Billman, Thomas ; Brandizzi, Federica ; Stapleton, Ann E. ( July 2019 , Scientific Reports)

   Plants are increasingly exposed to high temperatures, which can cause accumulation of unfolded protein in the endoplasmic reticulum (ER). This condition, known as ER stress, evokes the unfolded protein response (UPR), a cytoprotective signaling pathway. One important branch of the UPR is regulated by splicing of bZIP60 mRNA by the IRE1 stress sensor. There is increasing evidence that commercial plant growth regulators may protect against abiotic stressors including heat stress and drought, but there is very little mechanistic information about these effects or about the regulatory pathways involved. We evaluated evidence in the B73 Zea mays inbred for differences in the activity of the UPR between permissive and elevated temperature in conjunction with plant growth regulator application. Treatment with elevated temperature and plant growth regulators increased UPR activation, as assessed by an increase in splicing of the mRNA of the IRE1 target bZIP60 following paclobutrazol treatment. We propose that plant growth regulator treatment induces bZIP60 mRNA splicing which ‘primes’ plants for rapid adaptive response to subsequent endoplasmic reticulum-stress inducing conditions.

    

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4. Fitness and functional landscapes of the E. coli RNase III gene rnc

   https://doi.org/10.1093/molbev/msad047  

   Weeks, Ryan ; Ostermeier, Marc ( February 2023 , Molecular Biology and Evolution)

   Echave, Julian (Ed.)

   Abstract How protein properties such as protein activity and protein essentiality affect the distribution of fitness effects (DFE) of mutations are important questions in protein evolution. Deep mutational scanning studies typically measure the effects of a comprehensive set of mutations on either protein activity or fitness. Our understanding of the underpinnings of the DFE would be enhanced by a comprehensive study of both for the same gene. Here, we compared the fitness effects and in vivo protein activity effects of ∼4,500 missense mutations in the E. coli rnc gene. This gene encodes RNase III, a global regulator enzyme that cleaves diverse RNA substrates including precursor ribosomal RNA and various mRNAs including its own 5’ untranslated region (5’UTR). We find that RNase III’s ability to cleave dsRNA is the most important determinant of the fitness effects of rnc mutations. The DFE of RNase III was bimodal, with mutations centered around neutral and deleterious effects, consistent with previously reported DFE’s of enzymes with a singular physiological role. Fitness was buffered to small effects on RNase III activity. The enzyme’s RNase III domain (RIIID), which contains the RNase III signature motif and all active site residues, was more sensitive to mutation than its dsRNA binding domain (dsRBD), which is responsible for recognition and binding to dsRNA. Differential effects on fitness and functional scores for mutations at highly conserved residues G97, G99, and F188 suggest that these positions may be important for RNase III cleavage specificity. 

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5. The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

   https://doi.org/10.1371/journal.pbio.3001569  

   Yoon, Jimin ; Nekongo, Emmanuel E. ; Patrick, Jessica E. ; Hui, Tiffani ; Phillips, Angela M. ; Ponomarenko, Anna I. ; Hendel, Samuel J. ; Sebastian, Rebecca M. ; Zhang, Yu Meng ; Butty, Vincent L. ; et al ( February 2022 , PLOS Biology)

   Malik, Harmit S. (Ed.)

   The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation. 

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