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Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA-protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG-PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L's role., (© 2024 Riggs et al.)

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.

The compartmentalization of cellular function is achieved largely through the existence of membrane-bound organelles. However, recent work suggests a novel mechanism of compartmentalization mediated by membraneless structures that have liquid droplet-like properties and arise through phase separation. Cytoplasmic stress granules (SGs) are the best characterized and are induced by various stressors including arsenite, heat shock, and glucose deprivation. Current models suggest that SGs play an important role in protein homeostasis by mediating reversible translation attenuation. Protein phosphatase-1 (PP1) is a central cellular regulator responsible for most serine/threonine dephosphorylation. Here, we show that upon arsenite stress, PP1's catalytic subunit Glc7 relocalizes to punctate cytoplasmic granules. This altered localization requires PP1's recently described maturation pathway mediated by the multifunctional ATPase Cdc48 and PP1's regulatory subunit Ypi1. Glc7 relocalization is mediated by its regulatory subunit Reg1 and its target Snf1, the AMP-dependent protein kinase. Surprisingly, Glc7 granules are highly specific to arsenite and appear distinct from canonical SGs. Arsenite induces potent translational inhibition, and translational recovery is strongly dependent on Glc7, but independent of Glc7's well-established role in regulating eIF2α. These results suggest a novel form of stress-induced cytoplasmic granule and a new mode of translational control by Glc7., (© 2021 Federation of European Biochemical Societies.)

Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3°C survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. As protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold acclimation would evoke preparatory changes in protein expression to support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20°C (warm) or 3°C (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis. The relative abundance of 1316 identified proteins was compared between temperatures and developmental stages. The effect of cold acclimation on the cardiac proteome was only evident in the context of an interaction with life stage, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope with age that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of the life stage- and temperature-induced changes to the cardiac proteome, including reduced Complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stage-specific anoxia tolerance in turtles., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Stress granules (SGs) and processing bodies (PBs) are membraneless ribonucleoprotein-based cellular compartments that assemble in response to stress. SGs and PBs form through liquid-liquid phase separation that is driven by high local concentrations of key proteins and RNAs, both of which dynamically shuttle between the granules and the cytoplasm. SGs uniquely contain certain translation initiation factors and PBs are uniquely enriched with factors related to mRNA degradation and decay, although recent analyses reveal much broader protein commonality between these granules. Despite detailed knowledge of their composition and dynamics, the function of SGs and PBs remains poorly understood. Both, however, contain mRNAs, implicating their assembly in the regulation of RNA metabolism. SGs may also serve as hubs that rewire signaling events during stress. By contrast, PBs may constitute RNA storage centers, independent of mRNA decay. The aberrant assembly or disassembly of these granules has pathological implications in cancer, viral infection and neurodegeneration. Here, we review the current concepts regarding the formation, composition, dynamics, function and involvement in disease of SGs and PBs., (© 2020. Published by The Company of Biologists Ltd.)

Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrate. Annual killifish inhabit ephemeral ponds, producing drought and anoxia-tolerant embryos, which allows the species to persist generation after generation. Anoxia tolerance and physiology vary by developmental stage, creating a unique opportunity for comparative study within the species. A recent study of small ncRNA expression in A. limnaeus embryos in response to anoxia and aerobic recovery revealed small ncRNAs with expression patterns that suggest a role in supporting anoxia tolerance. MitosRNAs, small ncRNAs derived from the mitochondrial genome, emerged as an interesting group of these sequences. MitosRNAs derived from mitochondrial tRNAs were differentially expressed in developing embryos and isolated cells exhibiting extreme anoxia tolerance. In this study we focus on expression of mitosRNAs derived from tRNA-cysteine, and their subcellular and organismal localization in order to consider possible function. These tRNA-cys mitosRNAs appear enriched in the mitochondria, particularly near the nucleus, and also appear to be present in the cytoplasm. We provide evidence that mitosRNAs are generated in the mitochondria in response to anoxia, though the precise mechanism of biosynthesis remains unclear. MitosRNAs derived from tRNA-cys localize to numerous tissues, and increase in the anterior brain during anoxia. We hypothesize that these RNAs may play a role in regulating gene expression that supports extreme anoxia tolerance.

Most animal cells rely on aerobic metabolism for survival and are damaged or die within minutes without oxygen. Embryos of the annual killifish Austrofundulus limnaeus, however, survive months without oxygen. Determining how their cells survive without oxygen has the potential to revolutionize our understanding of the cellular mechanisms supporting vertebrate anoxia tolerance and the evolution of such tolerance. Therefore, we aimed to establish and characterize an anoxia-tolerant cell line from A. limnaeus for investigating mechanisms of vertebrate anoxia tolerance. The PSU-AL-WS40NE cell line of neuroepithelial identity was established from embryonic tissue of A. limnaeus using a tissue explant. The cells can survive for at least 49 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for two mammalian cell lines. PSU-AL-WS40NE cells accumulate lactate during anoxia, indicating use of common metabolic pathways for anaerobic metabolism. Additionally, they express many of the same small noncoding RNAs that are stress-responsive in whole embryos of A. limnaeus and mammalian cells, as well as anoxia-responsive small noncoding RNAs derived from the mitochondrial genome (mitosRNAs). The establishment of the cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus , the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark ( Hemiscyllium ocellatum ), crucian carp ( Carassius carassius ), western painted turtle ( Chrysemys picta bellii ), and leopard frog ( Rana pipiens ) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia. Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia. Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.

Background: The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in northern Venezuela, South America, and is an emerging extremophile model for vertebrate diapause, stress tolerance, and evolution. Embryos of A. limnaeus regularly experience extended periods of desiccation and anoxia as a part of their natural history and have unique metabolic and developmental adaptations. Currently, there are limited genomic resources available for gene expression and evolutionary studies that can take advantage of A. limnaeus as a unique model system., Results: We describe the first draft genome sequence of A. limnaeus. The genome was assembled de novo using a merged assembly strategy and was annotated using the NCBI Eukaryotic Annotation Pipeline. We show that the assembled genome has a high degree of completeness in genic regions that is on par with several other teleost genomes. Using RNA-seq and phylogenetic-based approaches, we identify several candidate genes that may be important for embryonic stress tolerance and post-diapause development in A. limnaeus. Several of these genes include heat shock proteins that have unique expression patterns in A. limnaeus embryos and at least one of these may be under positive selection., Conclusion: The A. limnaeus genome is the first South American annual killifish genome made publicly available. This genome will be a valuable resource for comparative genomics to determine the genetic and evolutionary mechanisms that support the unique biology of annual killifishes. In a broader context, this genome will be a valuable tool for exploring genome-environment interactions and their impacts on vertebrate physiology and evolution.

Background: Austrofundulus limnaeus is an annual killifish from the Maracaibo basin of Venezuela. Annual killifishes are unique among vertebrates in their ability to enter into a state of dormancy at up to three distinct developmental stages termed diapause I, II, and III. These embryos are tolerant of a wide variety of environmental stresses and develop relatively slowly compared with nonannual fishes., Results: These traits make them an excellent model for research on interactions between the genome and the environment during development, and an excellent choice for developmental biology laboratories. Furthermore, A. limnaeus is relatively easy to maintain in a laboratory setting and has a high fecundity, making it an excellent candidate as an emerging model for studies of development, and for defining the limits of developmental buffering in vertebrates., Conclusions: This study reports for the first time on the detailed development of A. limnaeus and provides a photographic and illustrated atlas of embryos on the two developmental trajectories possible in this species. Developmental Dynamics 246:779-801, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists., (© 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.)

Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance., (Copyright © 2017 the American Physiological Society.)

This study examined juvenile justice-involved youths' understanding and appreciation of the Miranda warnings' rights to silence and legal counsel using the Miranda Rights Comprehension Instruments (Goldstein, Zelle, & Grisso, 2012). It also examined the relationships between totality of circumstances factors and understanding and appreciation of rights. Data were collected from 183 youths (140 boys) in pre- and postadjudication facilities in 2 states. Overall, youths demonstrated greater difficulty on measures of appreciation than understanding, with particular deficits in their abilities to comprehend the abstract concept of the right to silence. Results varied slightly by instrument, highlighting the importance of a multimodal assessment of these complex abilities. Examination of totality of circumstances factors identified relationships between some factors (e.g., age, verbal IQ, academic achievement) and Miranda comprehension, but revealed that other factors (e.g., gender, number of previous arrests) were not significantly related to Miranda understanding or appreciation. The findings support a nuanced conceptualization of Miranda rights comprehension that acknowledges the complexity of understanding and appreciating the warnings. Empirical analyses also support the continued use of some totality of circumstances factors and abandonment of others. Findings underscore the necessity of multimodal assessment and interpretation when conducting capacity to waive Miranda rights evaluations., ((c) 2015 APA, all rights reserved).)

This article describes the psychometric properties of the Miranda Rights Comprehension Instruments, the revised version of Grisso's Miranda instruments. The original instruments demonstrated good reliability and validity in a normative sample. The revised instruments updated the content of the original instruments and were administered to a sample of 183 youth in pre- and postadjudication facilities. Analyses were conducted to establish the psychometric properties of the revised instruments and included similar analyses to those conducted by Grisso, as well as additional calculations (e.g., standard errors of measurement, intraclass correlation coefficients, Kappa coefficients). Results revealed sound psychometric properties, similar to those observed for the original instruments.