Highlights d SG assembly is driven by the collective interactions of a core protein-RNA network d The central node G3BP encodes a molecular switch that regulates RNA-dependent LLPS d Interplay between 3 distinct IDRs in G3BP tunes the intrinsic propensity for LLPS d Extrinsic factors regulate SG assembly through positive or negative cooperativity

SummaryLiquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in eukaryotic cells in response to a variety of stressors and are normally disassembled after stress is removed. The composition and assembly of stress granules is well understood, but little is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Using cultured human cells, we found that stress granule disassembly was context-dependent: Specifically in the setting of heat shock, disassembly required ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. We found that ubiquitinated G3BP1 interacted with the endoplasmic reticulum–associated protein FAF2, which engaged the ubiquitin-dependent segregase p97/VCP (valosin-containing protein). Thus, targeting of G3BP1 weakened the stress granule–specific interaction network, resulting in granule disassembly.

Mutations in the PFN1 gene encoding profilin 1 are a rare cause of familial amyotrophic lateral sclerosis (ALS). Profilin 1 is a well studied actin-binding protein but how PFN1 mutations cause ALS is unknown. The budding yeast, Saccharomyces cerevisiae, has one PFN1 ortholog. We expressed the ALS-linked profilin 1 mutant proteins in yeast, demonstrating a loss of protein stability and failure to restore growth to profilin mutant cells, without exhibiting gain-of-function toxicity. This model provides for simple and rapid screening of novel ALS-linked PFN1 variants. To gain insight into potential novel roles for profilin 1, we performed an unbiased, genome-wide synthetic lethal screen with yeast cells lacking profilin (pfy1⌬). Unexpectedly, deletion of several stress granule and processing body genes, including pbp1⌬, were found to be synthetic lethal with pfy1⌬. Mutations in ATXN2, the human ortholog of PBP1, are a known ALS genetic risk factor and ataxin 2 is a stress granule component in mammalian cells. Given this genetic interaction and recent evidence linking stress granule dynamics to ALS pathogenesis, we hypothesized that profilin 1 might also associate with stress granules. Here we report that profilin 1 and related protein profilin 2 are novel stress granule-associated proteins in mouse primary cortical neurons and in human cell lines and that ALS-linked mutations in profilin 1 alter stress granule dynamics, providing further evidence for the potential role of stress granules in ALS pathogenesis.

In the original Supplemental Information for this Article, the same image was used for both Figure S4 and Figure S5. This has been corrected in the Supplemental Information online.

Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in response to a variety of stressors and are normally disassembled after stress is removed. Whereas the composition of stress granules and the mechanisms underlying their assembly have been extensively studied, far less is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases. Here we report that stress granule disassembly is context-dependent and, in the setting of heat shock, requires ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. Ubiquitinated G3BP1 interacts with the ER-resident protein FAF2, which engages the ubiquitin-dependent segregase p97/VCP. Targeting G3BP1 enables the stress granule-specific interaction network to fall below the percolation threshold for phase separation, which causes disassembly.One Sentence SummaryUbiquitination of G3BP1 mediates FAF2- and p97/VCP-dependent disassembly of heat-induced stress granules

The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.