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 ...

SUMMARY The low complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS) and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state, and released for elongation following phosphorylation of the CTD.

Summary Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding, and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.

SUMMARY Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein.

Our paper identified nuclear proteins likely harboring disordered low-complexity sequences via precipitation by b-isox microcrystals. In Table S2, we ranked 580 nuclear proteins isolated in this manner and indicated that they were ordered according to the density of spectral counts. It has come to our attention that the proteins in this table are ordered by the relative density of [G/S]Y[G/S] triplet repeats rather than by spectral counts. This error affects the following sentence in the text of the Results section: ''Among the 580 mammalian proteins selectively precipitated by b-isox microcrystals, TAF15 registered the second highest number of spectral counts, and the largest subunit of RNA polymerase II registered the third highest number of spectral counts (Table S2).'' This is because the named positions had been based on ranking by triplet repeat density. We now provide with the article online the correctly ordered Table S2 (by spectral counts instead of triplet repeat density), and the affected sentence has now been changed to indicate the ranking positions of these proteins when ordered by spectral counts, such that TAF15 is 23 rd on the list and the largest subunit of RNA polymerase is 46 th. All proteins on the list are well above the false discovery rate, and the fact that both TAF15 and the largest subunit of RNA polymerase II are close to the very top of the list means that these adjustments do not alter any results or conclusions presented in the paper. We note that Table S3, which presents the yeast nuclear proteins precipitated by b-isox microcrystals, was correctly ordered by density of spectral counts as indicated. We wish to thank David Trudgian, a computational scientist in our Mass Sepctrometry Shared Resource Core, for pointing out the inconsistency in the organization and annotation of the original Table S2.