SUMMARY Stress granules are mRNA-protein granules that form when translation initiation is limited and are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures referred to as cores within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions, links between stress granules and human diseases, and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently; with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

SUMMARY Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-separated liquids organized by multivalent interactions among proteins and RNA molecules. Although many components of various cellular bodies are known, general principles that define body composition are lacking. We modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold molecules form phase-separated liquids that concentrate low valency client proteins. Clients partition differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. Composition can switch rapidly through changes in scaffold concentration or valency. Natural PML NBs and P bodies show analogous partitioning behavior, suggesting how their compositions could be controlled by levels of PML SUMOylation or cellular mRNA concentration, respectively. The data suggest a conceptual framework for considering the composition and control thereof of cellular bodies assembled through heterotypic multivalent interactions.

SUMMARY Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here we describe the stress granule transcriptome of yeast and mammalian cells through RNA-Seq analysis of purified stress granule cores and smFISH validation. While essentially every mRNA, and some ncRNAs, can be targeted to stress granules, the targeting efficiency varies from <1% to >95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-Seq analysis by smFISH reveals only 10% of bulk mRNA molecules accumulate in mammalian stress granules, and only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest stress granules may not represent a specific biological program of mRNP assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes.

Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.DOI: http://dx.doi.org/10.7554/eLife.18413.001

Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNPs (mRNA protein complexes) is essential to understanding these processes. In a global survey of S. cerevisiae mRNA binding proteins we identified 120 proteins that cross-link to mRNA, including 66 new mRNA binding proteins. These include kinases, RNA modification enzymes, metabolic enzymes, and tRNA and rRNA metabolism factors. These proteins show dynamic subcellular localization during stress, including assembly into stress granules and P-bodies (Processing-bodies). CLIP (cross-linking and immunoprecipitation) analyses of the P-body components Pat1, Lsm1, Dhh1 and Sbp1 identified sites of interaction on specific mRNAs revealing positional binding preferences and co-assembly preferences. Taken together, this work defines the major yeast mRNP proteins, reveals widespread changes in their subcellular location during stress, and begins to define assembly rules for P-body mRNPs.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which includes Crohn's disease (CD) and ulcerative colitis (UC). These diseases have become important health problems. Medical therapy for IBD has advanced dramatically in the last decade with the introduction of targeted biologic therapies, the optimization of older therapies, including rugs such as immunomodulators and 5-aminosalicylic acid (5-ASA), and a better understanding of the mucosal immune system and the genetics involved in the pathogenesis of IBD. The goal of IBD therapy is to induce and maintain remission. The current treatment paradigm involves a step-up approach, moving to aggressive, powerful therapies only when milder therapies with fewer potential side effects fail or when patients declare themselves to have an aggressive disease. This review focuses on the current treatments for inflammatory bowel disease.

Stress granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian stress granules are comprised of two structures – an unstable shell and more stable cores. Herein we describe methodology for isolation of stress granule cores from both yeast and mammalian cells. The protocol consists of first enriching for stress granule cores using centrifugation and then further purifying stress granule cores using immunoprecipitation. The stress granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.

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