Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

SUMMARY Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.

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Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.

Many RNA regulatory proteins controlling pre-mRNA splicing contain serine:arginine (SR) repeats. Here we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels, but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9ORF72 gene altered in neurodegenerative disease encode GR N and PR N repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.Among familial causes of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), between 25 and 40% of cases are attributed to a repeat expansion in a gene designated C9ORF72. The hexa-nucleotide repeat sequence GGGGCC normally present in 2 to 23 copies is expanded in affected patients to 700 to 1,600 copies (1, 2). The pattern of genetic inheritance of the C9ORF72 repeat expansion is dominant, and multiple lines of evidence suggest that the repeat expansion causes disease. Two theories have been advanced to explain repeat-generated toxicity. First, in situ hybridization assays have identified nuclear dots containing either sense or anti-sense repeat transcripts (3-5), leading to the idea that the nuclear-retained RNAs might themselves be toxic. More recently, equally clear evidence has been generated showing that both the sense and anti-sense transcripts of the * Corresponding author. steven.mcknight@utsouthwestern.edu. SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.1254917/DC1 Materials and Methods Figs. S1 to S6 Table S1 References (29)(30)(31)(32)(33)(34) HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript GGGGCC repeats associated with C9ORF72 can be translated in an ATG-independent manner known as repeat associated non-ATG (RAN) translation (6). Depending upon reading frame, the sense transcript of the repeats can be translated into glycine:alanine (GA N ), glycine:proline (GP N ), or glycine:arginine (GR N ) polymers. RAN translation of the anti-sense transcript of the GGGGCC repeats of C9ORF72 lead to the production of proline:alanine (PA N ), proline:glycine (PG N ) or proline:arginine (PR N ) polymers. These repeat-encoded polymers are expressed in disease tissue (5, 7-9). The disordered and hydrophobic nature of these polymers, at least the GA N , GP N , and PA N versions, properly predicted that they would aggregate into distinct foci within affected cells (5, 9). Another plausible explanation for repeat-generated toxicity ...