RESUMEN
The RNA-binding protein FUS (Fused in Sarcoma) mediates phase separation in biomolecular condensates and functions in transcription by clustering with RNA polymerase II. Specific contact residues and interaction modes formed by FUS and the C-terminal heptad repeats of RNA polymerase II (CTD) have been suggested but not probed directly. Here we show how RGG domains contribute to phase separation with the FUS N-terminal low-complexity domain (SYGQ LC) and RNA polymerase II CTD. Using NMR spectroscopy and molecular simulations, we demonstrate that many residue types, not solely arginine-tyrosine pairs, form condensed-phase contacts via several interaction modes including, but not only sp2-π and cation-π interactions. In phases also containing RNA polymerase II CTD, many residue types form contacts, including both cation-π and hydrogen-bonding interactions formed by the conserved human CTD lysines. Hence, our data suggest a surprisingly broad array of residue types and modes explain co-phase separation of FUS and RNA polymerase II.
Asunto(s)
Condensados Biomoleculares/fisiología , ARN Polimerasa II/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Comunicación Celular/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Enlace de Hidrógeno , Lisina/química , Espectroscopía de Resonancia Magnética , Dominios Proteicos/fisiología , Transcripción Genética/genéticaRESUMEN
Many cancer-causing chromosomal translocations result in transactivating protein products encoding FET family (FUS, EWSR1, TAF15) low-complexity (LC) domains fused to a DNA binding domain from one of several transcription factors. Recent work demonstrates that higher-order assemblies of FET LC domains bind the carboxy-terminal domain of the large subunit of RNA polymerase II (RNA pol II CTD), suggesting FET oncoproteins may mediate aberrant transcriptional activation by recruiting RNA polymerase II to promoters of target genes. Here we use nuclear magnetic resonance (NMR) spectroscopy and hydrogel fluorescence microscopy localization and fluorescence recovery after photobleaching to visualize atomic details of a model of this process, interactions of RNA pol II CTD with high-molecular weight TAF15 LC assemblies. We report NMR resonance assignments of the intact degenerate repeat half of human RNA pol II CTD alone and verify its predominant intrinsic disorder by molecular simulation. By measuring NMR spin relaxation and dark-state exchange saturation transfer, we characterize the interaction of RNA pol II CTD with amyloid-like hydrogel fibrils of TAF15 and hnRNP A2 LC domains and observe that heptads far from the acidic C-terminal tail of RNA pol II CTD bind TAF15 fibrils most avidly. Mutation of CTD lysines in heptad position 7 to consensus serines reduced the overall level of TAF15 fibril binding, suggesting that electrostatic interactions contribute to complex formation. Conversely, mutations of position 7 asparagine residues and truncation of the acidic tail had little effect. Thus, weak, multivalent interactions between TAF15 fibrils and heptads throughout RNA pol II CTD collectively mediate complex formation.
Asunto(s)
ARN Polimerasa II/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Transcripción Genética , Translocación Genética/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Lisina/química , Lisina/genética , Espectroscopía de Resonancia Magnética , Complejos Multiproteicos , Mutación , Neoplasias/genética , Neoplasias/patología , Regiones Promotoras Genéticas , Unión Proteica , Dominios Proteicos/genética , ARN Polimerasa II/química , Factores Asociados con la Proteína de Unión a TATA/químicaRESUMEN
Neuronal inclusions of aggregated RNA-binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation-prone, yeast prion-like, low sequence-complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in-cell phosphorylation sites across FUS LC We show that both phosphorylation and phosphomimetic variants reduce its aggregation-prone/prion-like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self-interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity. Hence, post-translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/patología , Línea Celular , Demencia Frontotemporal/patología , Humanos , Espectroscopía de Resonancia Magnética , Fosforilación , Agregación Patológica de Proteínas , Conformación Proteica , Proteína FUS de Unión a ARN/químicaRESUMEN
Liquid-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.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Arginina/metabolismo , Gránulos Citoplasmáticos/metabolismo , Dipéptidos/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Arginina/química , Proteína C9orf72 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Gránulos Citoplasmáticos/patología , ADN Helicasas , Dipéptidos/química , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Proteínas Intrínsecamente Desordenadas/química , Gotas Lipídicas/metabolismo , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa , Dominios Proteicos , Proteínas/química , ARN/metabolismo , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Factores de Tiempo , TransfecciónRESUMEN
Phase-separated states of proteins underlie ribonucleoprotein (RNP) granules and nuclear RNA-binding protein assemblies that may nucleate protein inclusions associated with neurodegenerative diseases. We report that the N-terminal low-complexity domain of the RNA-binding protein Fused in Sarcoma (FUS LC) is structurally disordered and forms a liquid-like phase-separated state resembling RNP granules. This state directly binds the C-terminal domain of RNA polymerase II. Phase-separated FUS lacks static structures as probed by fluorescence microscopy, indicating they are distinct from both protein inclusions and hydrogels. We use solution nuclear magnetic resonance spectroscopy to directly probe the dynamic architecture within FUS liquid phase-separated assemblies. Importantly, we find that FUS LC retains disordered secondary structure even in the liquid phase-separated state. Therefore, we propose that disordered protein granules, even those made of aggregation-prone prion-like domains, are dynamic and disordered molecular assemblies with transiently formed protein-protein contacts.