SUMMARY Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia and multisystem proteinopathy - diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. Here we demonstrate that the RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties, and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease.

SummaryAlgorithms designed to identify canonical yeast prions predict that ~250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbor a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here, we define pathogenic mutations in PrLDs of hnRNPA2/B1 and hnRNPA1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and a case of familial ALS. Wild-type hnRNPA2 and hnRNPA1 display an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Importantly, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant ‘steric zipper’ motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs must be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

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

Summary Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

GGGGCC (G4C2) repeat expansion in a noncoding region of C9ORF72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1,2. The basis for pathogenesis is unknown. To capture the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2 repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein (GFP) to detect repeat-associated non-AUG (RAN) translation. These transgenic animals show dosage-dependent, repeat length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins as observed in patients. This model was used in a large-scale, unbiased genetic screen ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC) as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged iPSC-derived neurons from C9ORF72 patients. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.