Proteasome granular localization is regulated through mitochondrial respiration and kinase signaling

In yeast, proteasomes are enriched in cell nuclei where they execute important cellular functions. Nutrient-stress can change this localization indicating proteasomes respond to the cell's metabolic state. However, the signals that connect these processes remain poorly understood. Carbon starvation triggers a reversible translocation of proteasomes to cytosolic condensates known as proteasome storage granules (PSGs). Surprisingly, we observed strongly reduced PSG levels when cells had active cellular respiration prior to starvation. This suggests the mitochondrial activity of cells is a determining factor in the response of proteasomes to carbon starvation. Consistent with this, upon inhibition of mitochondrial function we observed proteasomes relocalize to granules. These links between proteasomes and metabolism involve specific signaling pathways, as we identified a MAP kinase cascade that is critical to the formation of proteasome granules after respiratory growth but not following glycolytic growth. Furthermore, the yeast homolog of AMP kinase, Snf1, is important for proteasome granule formation induced by mitochondrial inhibitors, while dispensable for granule formation following carbon starvation. We propose a model where mitochondrial activity promotes proteasome nuclear localization.

The Transcriptional Response to Oxidative Stress is Independent of Stress-Granule Formation

Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3-hours predominantly resulted in induction or upregulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for chaperones, including HSP70s, small heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules

Insulin is a key regulator of human metabolism, and its dysfunction leads to diseases such as type 2 diabetes. It remains unknown how proinsulin is targeted from the trans-Golgi network (TGN) to secretory storage granules as no cargo receptor has been identified. Chromogranin proteins (CGs) are central regulators of granule biosynthesis, and it was proposed that their aggregation is critical for this process. However, the molecular mechanism by which these molecules facilitate sorting at the TGN is poorly understood. Here, we show that CGs undergo liquid-liquid phase separation (LLPS) at low pH independently of divalent cations, such as calcium. Liquid CG condensates, but not aggregates, recruit and sort proinsulin and other granule destined cargo molecules towards secretory granules. Cargo selectivity is independent of sequence or structural elements but is based on the size and concentration of the client molecules at the TGN. Finally, electrostatic interactions and the N-terminal intrinsically disordered domain of chromogranin B facilitate LLPS and are critical for granule formation. We propose that phase-separated CGs act as a cargo sponge within the TGN lumen, gathering soluble client proteins into the condensate independently of specific sequence or structural elements, facilitating receptor-independent sorting. These findings challenge the canonical TGN sorting models and provide insights into granule biosynthesis in insulin-secreting beta cells.

Gut Metabolite Trimethylamine N-Oxide Protects INS-1 β-Cell and Rat Islet Function under Diabetic Glucolipotoxic Conditions

Serum accumulation of the gut microbial metabolite trimethylamine N-oxide (TMAO) is associated with high caloric intake and type 2 diabetes (T2D). Impaired pancreatic β-cell function is a hallmark of diet-induced T2D, which is linked to hyperglycemia and hyperlipidemia. While TMAO production via the gut microbiome-liver axis is well defined, its molecular effects on metabolic tissues are unclear, since studies in various tissues show deleterious and beneficial TMAO effects. We investigated the molecular effects of TMAO on functional β-cell mass. We hypothesized that TMAO may damage functional β-cell mass by inhibiting β-cell viability, survival, proliferation, or function to promote T2D pathogenesis. We treated INS-1 832/13 β-cells and primary rat islets with physiological TMAO concentrations and compared functional β-cell mass under healthy standard cell culture (SCC) and T2D-like glucolipotoxic (GLT) conditions. GLT significantly impeded β-cell mass and function by inducing oxidative and endoplasmic reticulum (ER) stress. TMAO normalized GLT-mediated damage in β-cells and primary islet function. Acute 40µM TMAO recovered insulin production, insulin granule formation, and insulin secretion by upregulating the IRE1α unfolded protein response to GLT-induced ER and oxidative stress. These novel results demonstrate that TMAO protects β-cell function and suggest that TMAO may play a beneficial molecular role in diet-induced T2D conditions.

Cooperativity and Interdependency between RNA Structure and RNA–RNA Interactions

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions.

Polyadenylated RNA and RNA-Binding Proteins Exhibit Unique Response to Hyperosmotic Stress

Stress granule formation is a complex and rapidly evolving process that significantly disrupts cellular metabolism in response to a variety of cellular stressors. Recently, it has become evident that different chemical stressors lead to the formation of compositionally distinct stress granules. However, it is unclear which proteins are required for the formation of stress granules under different conditions. In addition, the effect of various stressors on polyadenylated RNA metabolism remains enigmatic. Here, we demonstrate that G3BP1/2, which are common stress granule components, are not required for the formation of stress granules specifically during osmotic stress induced by sorbitol and related polyols. Furthermore, sorbitol-induced osmotic stress leads to significant depletion of nuclear polyadenylated RNA, a process that we demonstrate is dependent on active mRNA export, as well as cytoplasmic and subnuclear shifts in the presence of many nuclear RNA-binding proteins. We assessed the function of multiple shifted RBPs and found that hnRNP U, but not TDP-43 or hnRNP I, exhibit reduced function following this cytoplasmic shift. Finally, we observe that multiple stress pathways lead to a significant reduction in transcription, providing a possible explanation for our inability to observe loss of TDP-43 or hnRNP I function. Overall, we identify unique outcomes following osmotic stress that provide important insight into the regulation of RNA-binding protein localization and function.

PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential “prion-like” domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59’s localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation.

U2AF1 Mutations Enhance Stress Granule Response in Myeloid Malignancies

Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies. We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding. Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components. Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. Disclosures Hunck: Boehringer Ingelheim: Other: Fellowship.