Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 109
Filtrar
1.
Nat Commun ; 12(1): 1809, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753744

RESUMEN

Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.


Asunto(s)
Proteínas Fluorescentes Verdes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Multimerización de Proteína , Proteínas/química , Animales , Células COS , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Proteínas/genética , Proteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo
2.
Nat Commun ; 12(1): 1085, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33597515

RESUMEN

Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.


Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Transición de Fase , Proteínas/química , Electricidad Estática , Animales , Anexinas/química , Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Humanos , Proteína FUS de Unión a ARN/química , Factores de Transcripción SOXB1/química , Células Sf9 , Spodoptera , Factores de Transcripción/química
3.
Nat Commun ; 11(1): 6341, 2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33311468

RESUMEN

Mutations in the RNA-binding protein Fused in Sarcoma (FUS) cause early-onset amyotrophic lateral sclerosis (ALS). However, a detailed understanding of central RNA targets of FUS and their implications for disease remain elusive. Here, we use a unique blend of crosslinking and immunoprecipitation (CLIP) and NMR spectroscopy to identify and characterise physiological and pathological RNA targets of FUS. We find that U1 snRNA is the primary RNA target of FUS via its interaction with stem-loop 3 and provide atomic details of this RNA-mediated mode of interaction with the U1 snRNP. Furthermore, we show that ALS-associated FUS aberrantly contacts U1 snRNA at the Sm site with its zinc finger and traps snRNP biogenesis intermediates in human and murine motor neurons. Altogether, we present molecular insights into a FUS toxic gain-of-function involving direct and aberrant RNA-binding and strengthen the link between two motor neuron diseases, ALS and spinal muscular atrophy (SMA).


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular , Predisposición Genética a la Enfermedad/genética , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Neuronas Motoras/metabolismo , Mutación , Dominios y Motivos de Interacción de Proteínas , ARN Nuclear Pequeño/química , Proteína FUS de Unión a ARN/química , Ribonucleoproteína Nuclear Pequeña U1/química
4.
Nat Commun ; 11(1): 5735, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33184287

RESUMEN

Protein domains without the usual distribution of amino acids, called low complexity (LC) domains, can be prone to self-assembly into amyloid-like fibrils. Self-assembly of LC domains that are nearly devoid of hydrophobic residues, such as the 214-residue LC domain of the RNA-binding protein FUS, is particularly intriguing from the biophysical perspective and is biomedically relevant due to its occurrence within neurons in amyotrophic lateral sclerosis, frontotemporal dementia, and other neurodegenerative diseases. We report a high-resolution molecular structural model for fibrils formed by the C-terminal half of the FUS LC domain (FUS-LC-C, residues 111-214), based on a density map with 2.62 Å resolution from cryo-electron microscopy (cryo-EM). In the FUS-LC-C fibril core, residues 112-150 adopt U-shaped conformations and form two subunits with in-register, parallel cross-ß structures, arranged with quasi-21 symmetry. All-atom molecular dynamics simulations indicate that the FUS-LC-C fibril core is stabilized by a plethora of hydrogen bonds involving sidechains of Gln, Asn, Ser, and Tyr residues, both along and transverse to the fibril growth direction, including diverse sidechain-to-backbone, sidechain-to-sidechain, and sidechain-to-water interactions. Nuclear magnetic resonance measurements additionally show that portions of disordered residues 151-214 remain highly dynamic in FUS-LC-C fibrils and that fibrils formed by the N-terminal half of the FUS LC domain (FUS-LC-N, residues 2-108) have the same core structure as fibrils formed by the full-length LC domain. These results contribute to our understanding of the molecular structural basis for amyloid formation by FUS and by LC domains in general.


Asunto(s)
Amiloide/química , Amiloide/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/metabolismo , Amiloide/genética , Amiloide/ultraestructura , Microscopía por Crioelectrón , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Estructura Molecular , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/ultraestructura , Análisis de Secuencia de Proteína
5.
Nat Struct Mol Biol ; 27(4): 363-372, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32231288

RESUMEN

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.


Asunto(s)
Proteínas de Choque Térmico HSP27/química , Chaperonas Moleculares/química , Proteína FUS de Unión a ARN/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/aislamiento & purificación , Humanos , Extracción Líquido-Líquido , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Fosforilación , Unión Proteica/genética , Dominios Proteicos/genética , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/aislamiento & purificación , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Estrés Fisiológico/genética
6.
Sci Rep ; 10(1): 2629, 2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-32060318

RESUMEN

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained. Here, the closed-to-open conformational change of TLS on binding pncRNA was implied by fluorescence resonance energy transfer. A small fragment (31 nucleotides) of the full-length pncRNA (602 nucleotides) was shown to be sufficient for the conformational change of TLS. Dissection of pncRNA identified the G-rich RNA sequence that is critical for the conformational change. The length of RNA was also revealed to be critical for the conformational change. Furthermore, it was demonstrated that the conformational change of TLS is caused by another target DNA and RNA, telomeric DNA and telomeric repeat-containing RNA. The conformational change of TLS on binding target RNA/DNA is suggested to be essential for biological functions.


Asunto(s)
ARN no Traducido/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Transferencia Resonante de Energía de Fluorescencia , Humanos , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , ARN no Traducido/química , Proteína FUS de Unión a ARN/química
7.
Biochemistry ; 59(4): 364-378, 2020 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-31895552

RESUMEN

In aqueous solutions, the 214-residue low-complexity domain of the FUS protein (FUS-LC) is known to undergo liquid-liquid phase separation and also to self-assemble into amyloid-like fibrils. In previous work based on solid state nuclear magnetic resonance (ssNMR) methods, a structural model for the FUS-LC fibril core was developed, showing that residues 39-95 form the fibril core. Unlike fibrils formed by amyloid-ß peptides, α-synuclein, and other amyloid-forming proteins, the FUS-LC core is largely devoid of purely hydrophobic amino acid side chains. Instead, the core-forming segment contains numerous hydroxyl-bearing residues, including 18 serines, six threonines, and eight tyrosines, suggesting that the FUS-LC fibril structure may be stabilized in part by inter-residue hydrogen bonds among side chain hydroxyl groups. Here we describe ssNMR measurements, performed on 2H,15N,13C-labeled FUS-LC fibrils, that provide new information about the interactions of hydroxyl-bearing residues with one another and with water. The ssNMR data support the involvement of specific serine, threonine, and tyrosine residues in hydrogen-bonding interactions. The data also reveal differences in hydrogen exchange rates with water for different side chain hydroxyl groups, providing information about solvent exposure and penetration of water into the FUS-LC fibril core.


Asunto(s)
Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/ultraestructura , Secuencia de Aminoácidos/genética , Amiloide/química , Péptidos beta-Amiloides/metabolismo , Proteínas Amiloidogénicas/metabolismo , Humanos , Hidrógeno/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína/fisiología , Proteína FUS de Unión a ARN/genética
8.
Hum Mol Genet ; 28(R2): R187-R196, 2019 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-31595953

RESUMEN

Recent work on the biophysics of proteins with low complexity, intrinsically disordered domains that have the capacity to form biological condensates has profoundly altered the concepts about the pathogenesis of inherited and sporadic neurodegenerative disorders associated with pathological accumulation of these proteins. In the present review, we use the FUS, TDP-43 and A11 proteins as examples to illustrate how missense mutations and aberrant post-translational modifications of these proteins cause amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD).


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Anexinas/genética , Proteínas de Unión al ADN/genética , Degeneración Lobar Frontotemporal/genética , Proteína FUS de Unión a ARN/química , Lóbulo Temporal/fisiopatología , Anexinas/química , Anexinas/metabolismo , Transporte Biológico/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Mutación Missense , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/química , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo
9.
Curr Opin Struct Biol ; 59: 134-142, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31479821

RESUMEN

RNA-binding proteins TDP-43 and FUS play essential roles in pre-mRNA splicing, localization, granule formation and other aspects of RNA metabolism. Both proteins are implicated in neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite their apparent similarities, each protein has unique structural characteristics. Here we present the current structural understanding of RNA-binding and self-association mechanisms. Both globular and intrinsically disordered domains contribute to RNA binding, each with different specificities, affinities and kinetics. Self-associating Prion-like domains in each protein form multivalent interactions and labile cross-ß structures. These interactions are modulated by distinctive additional domains including a globular oligomerization domain in TDP-43 and synergistic interactions with intrinsically disordered Arginine-Glycine rich domains in FUS. These insights contribute to a better understanding of native biological functions of TDP-43 and FUS and potential molecular pathways in neurodegenerative diseases.


Asunto(s)
Proteínas de Unión al ADN/química , Proteína FUS de Unión a ARN/química , ARN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , ARN/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo
10.
Nat Struct Mol Biol ; 26(7): 637-648, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31270472

RESUMEN

The low-complexity domain of the RNA-binding protein FUS (FUS LC) mediates liquid-liquid phase separation (LLPS), but the interactions between the repetitive SYGQ-rich sequence of FUS LC that stabilize the liquid phase are not known in detail. By combining NMR and Raman spectroscopy, mutagenesis, and molecular simulation, we demonstrate that heterogeneous interactions involving all residue types underlie LLPS of human FUS LC. We find no evidence that FUS LC adopts conformations with traditional secondary structure elements in the condensed phase; rather, it maintains conformational heterogeneity. We show that hydrogen bonding, π/sp2, and hydrophobic interactions all contribute to stabilizing LLPS of FUS LC. In addition to contributions from tyrosine residues, we find that glutamine residues also participate in contacts leading to LLPS of FUS LC. These results support a model in which FUS LC forms dynamic, multivalent interactions via multiple residue types and remains disordered in the densely packed liquid phase.


Asunto(s)
Proteína FUS de Unión a ARN/química , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Intrínsecamente Desordenadas/química , Modelos Moleculares , Transición de Fase , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína
11.
Commun Biol ; 2: 223, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31240261

RESUMEN

Adenosine triphosphate (ATP) provides energy for cellular processes but has recently been found to act also as a hydrotrope to maintain protein homeostasis. ATP bivalently binds the disordered domain of FUS containing the RG/RGG sequence motif and thereby affects FUS liquid-liquid phase separation. Here, using NMR spectroscopy and molecular docking studies, we report that ATP specifically binds also to the well-folded RRM domain of FUS at physiologically relevant concentrations and with the binding interface overlapping with that of its physiological ssDNA ligand. Importantly, although ATP has little effect on the thermodynamic stability of the RRM domain or its binding to ssDNA, ATP kinetically inhibits the RRM fibrillization that is critical for the gain of cytotoxicity associated with ALS and FTD. Our study provides a previously unappreciated mechanism for ATP to inhibit fibrillization by specific binding, and suggests that ATP may bind additional proteins other than the classic ATP-dependent enzymes.


Asunto(s)
Adenosina Trifosfato/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Adenosina Trifosfato/química , ADN de Cadena Simple , Humanos , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Enfermedades Neurodegenerativas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Proteína FUS de Unión a ARN/química , Termodinámica
12.
Cell Rep ; 27(6): 1809-1821.e5, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-31067465

RESUMEN

PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNA-rich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates.


Asunto(s)
Daño del ADN , Glicósido Hidrolasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Compartimento Celular , Activación Enzimática , Femenino , Células HeLa , Humanos , Peróxido de Hidrógeno/toxicidad , Modelos Biológicos , Fosforilación , Poli Adenosina Difosfato Ribosa/metabolismo , Dominios Proteicos , Proteína FUS de Unión a ARN/química
13.
Molecules ; 24(8)2019 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-31022909

RESUMEN

Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid-liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases.


Asunto(s)
Enfermedades Neurodegenerativas/genética , Agregación Patológica de Proteínas/genética , Proteína FUS de Unión a ARN/química , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Asparagina/química , Asparagina/genética , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/patología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Humanos , Enfermedades Neurodegenerativas/patología , Péptidos/química , Péptidos/genética , Priones/química , Priones/genética , Agregación Patológica de Proteínas/patología , Dominios Proteicos/genética , Proteína FUS de Unión a ARN/genética
14.
Sci Rep ; 9(1): 6171, 2019 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-30992467

RESUMEN

Aberrant expression, dysfunction and particularly aggregation of a group of RNA-binding proteins, including TDP-43, FUS and RBM45, are associated with neurological disorders. These three disease-linked RNA-binding proteins all contain at least one RNA recognition motif (RRM). However, it is not clear if these RRMs contribute to their aggregation-prone character. Here, we compare the biophysical and fibril formation properties of five RRMs from disease-linked RNA-binding proteins and five RRMs from non-disease-associated proteins to determine if disease-linked RRMs share specific features making them prone to self-assembly. We found that most of the disease-linked RRMs exhibit reversible thermal unfolding and refolding, and have a slightly lower average thermal melting point compared to that of normal RRMs. The full domain of TDP-43 RRM1 and FUS RRM, as well as the ß-peptides from these two RRMs, could self-assemble into fibril-like aggregates which are amyloids of parallel ß-sheets as verified by X-ray diffraction and FT-IR spectroscopy. Our results suggest that some disease-linked RRMs indeed play important roles in amyloid formation and shed light on why RNA-binding proteins with RRMs are frequently identified in the cellular inclusions of neurodegenerative diseases.


Asunto(s)
Amiloide/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Amiloide/química , Amiloide/ultraestructura , Proteínas de Unión al ADN/química , Humanos , Proteínas del Tejido Nervioso/química , Agregado de Proteínas , Desplegamiento Proteico , Motivo de Reconocimiento de ARN , Proteína FUS de Unión a ARN/química , Proteínas de Unión al ARN/química , Temperatura
15.
Mol Cell ; 73(3): 490-504.e6, 2019 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-30581145

RESUMEN

Fused in sarcoma (FUS) is an RNA binding protein involved in regulating many aspects of RNA processing and linked to several neurodegenerative diseases. Transcriptomics studies indicate that FUS binds a large variety of RNA motifs, suggesting that FUS RNA binding might be quite complex. Here, we present solution structures of FUS zinc finger (ZnF) and RNA recognition motif (RRM) domains bound to RNA. These structures show a bipartite binding mode of FUS comprising of sequence-specific recognition of a NGGU motif via the ZnF and an unusual shape recognition of a stem-loop RNA via the RRM. In addition, sequence-independent interactions via the RGG repeats significantly increase binding affinity and promote destabilization of structured RNA conformation, enabling additional binding. We further show that disruption of the RRM and ZnF domains abolishes FUS function in splicing. Altogether, our results rationalize why deciphering the RNA binding mode of FUS has been so challenging.


Asunto(s)
Proteína FUS de Unión a ARN/química , ARN/química , Sitios de Unión , Células HeLa , Humanos , Modelos Moleculares , Motivos de Nucleótidos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/genética , ARN/metabolismo , Motivo de Reconocimiento de ARN , Empalme del ARN , Estabilidad del ARN , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Relación Estructura-Actividad , Dedos de Zinc
16.
Biochemistry ; 57(51): 7021-7032, 2018 12 26.
Artículo en Inglés | MEDLINE | ID: mdl-30488693

RESUMEN

Purified recombinant FUsed in Sarcoma (FUS) assembles into an oligomeric state in an RNA-dependent manner to form large condensates. FUS condensates bind and concentrate the C-terminal domain of RNA polymerase II (RNA Pol II). We asked whether a granule in cells contained FUS and RNA Pol II as suggested by the binding of FUS condensates to the polymerase. We developed cross-linking protocols to recover protein particles containing FUS from cells and separated them by size exclusion chromatography. We found a significant fraction of RNA Pol II in large granules containing FUS with diameters of >50 nm or twice that of the RNA Pol II holoenzyme. Inhibition of transcription prevented the polymerase from associating with the granules. Altogether, we found physical evidence of granules containing FUS and RNA Pol II in cells that possess properties comparable to those of in vitro FUS condensates.


Asunto(s)
ARN Polimerasa II/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Reactivos de Enlaces Cruzados , Células HEK293 , Humanos , Microscopía Electrónica de Transmisión , Modelos Biológicos , Tamaño de la Partícula , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/química , ARN Polimerasa II/genética , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcripción Genética
17.
Mol Biol Cell ; 29(15): 1786-1797, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29897835

RESUMEN

FUS (fused in sarcoma) is an abundant, predominantly nuclear protein involved in RNA processing. Under various conditions, FUS functionally associates with RNA and other macromolecules to form distinct, reversible phase-separated liquid structures. Persistence of the phase-separated state and increased cytoplasmic localization are both hypothesized to predispose FUS to irreversible aggregation, which is a pathological hallmark of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. We previously showed that phosphorylation of FUS's prionlike domain suppressed phase separation and toxic aggregation, proportionally to the number of added phosphates. However, phosphorylation of FUS's prionlike domain was previously reported to promote its cytoplasmic localization, potentially favoring pathological behavior. Here we used mass spectrometry and human cell models to further identify phosphorylation sites within FUS's prionlike domain, specifically following DNA-damaging stress. In total, 28 putative sites have been identified, about half of which are DNA-dependent protein kinase (DNA-PK) consensus sites. Custom antibodies were developed to confirm the phosphorylation of two of these sites (Ser-26 and Ser-30). Both sites were usually phosphorylated in a subpopulation of cellular FUS following a variety of DNA-damaging stresses but not necessarily equally or simultaneously. Importantly, we found DNA-PK-dependent multiphosphorylation of FUS's prionlike domain does not cause cytoplasmic localization.


Asunto(s)
Núcleo Celular/metabolismo , Daño del ADN , Priones/química , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/metabolismo , Secuencia de Aminoácidos , Aminoglicósidos/farmacología , Línea Celular , Núcleo Celular/efectos de los fármacos , Proteína Quinasa Activada por ADN/metabolismo , Humanos , Fosforilación/efectos de los fármacos , Dominios Proteicos , Transporte de Proteínas/efectos de los fármacos
18.
Sci Rep ; 8(1): 7084, 2018 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-29728564

RESUMEN

TDP-43 and FUS are nuclear proteins with multiple functions in mRNA processing. They play key roles in ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia), where they are partially lost from the nucleus and aggregate in the cytoplasm of neurons and glial cells. Defects in nucleocytoplasmic transport contribute to this pathology, hence nuclear import of both proteins has been studied in detail. However, their nuclear export routes remain poorly characterized and it is unclear whether aberrant nuclear export contributes to TDP-43 or FUS pathology. Here we show that predicted nuclear export signals in TDP-43 and FUS are non-functional and that both proteins are exported independently of the export receptor CRM1/Exportin-1. Silencing of Exportin-5 or the mRNA export factor Aly/REF, as well as mutations that abrogate RNA-binding do not impair export of TDP-43 and FUS. However, artificially enlarging TDP-43 or FUS impairs their nuclear egress, suggesting that they could leave the nucleus by passive diffusion. Finally, we found that inhibition of transcription causes accelerated nuclear egress of TDP-43, suggesting that newly synthesized RNA retains TDP-43 in the nucleus, limiting its egress into the cytoplasm. Our findings implicate reduced nuclear retention as a possible factor contributing to mislocalization of TDP-43 in ALS/FTD.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Carioferinas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Unión al ADN/química , Humanos , Carioferinas/química , Unión Proteica , Señales de Clasificación de Proteína , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/química , Receptores Citoplasmáticos y Nucleares/química
19.
Nucleic Acids Res ; 46(12): 5894-5901, 2018 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-29800261

RESUMEN

The RGG domain, defined as closely spaced Arg-Gly-Gly repeats, is a DNA and RNA-binding domain in various nucleic acid-binding proteins. Translocated in liposarcoma (TLS), which is also called FUS, is a protein with three RGG domains, RGG1, RGG2 and RGG3. TLS/FUS binding to G-quadruplex telomere DNA and telomeric repeat-containing RNA depends especially on RGG3, comprising Arg-Gly-Gly repeats with proline- and arginine-rich regions. So far, however, only non-specific DNA and RNA binding of TLS/FUS purified with buffers containing urea and KCl have been reported. Here, we demonstrate that protein purification using a buffer with high concentrations of urea and KCl decreases the G-quadruplex binding abilities of TLS/FUS and RGG3, and disrupts the ß-spiral structure of RGG3. Moreover, the Arg-Gly-Gly repeat region in RGG3 by itself cannot form a stable ß-spiral structure that binds to the G-quadruplex, because the proline- and arginine-rich regions induce the ß-spiral structure and the G-quadruplex-binding ability of RGG3. Our findings suggest that the G-quadruplex-specific binding abilities of TLS/FUS require RGG3 with a ß-spiral structure stabilized by adjacent proline- and arginine-regions.


Asunto(s)
G-Cuádruplex , Proteína FUS de Unión a ARN/química , Arginina/análisis , Cloruro de Potasio , Prolina/análisis , Unión Proteica , Dominios Proteicos , Proteína FUS de Unión a ARN/aislamiento & purificación , Proteína FUS de Unión a ARN/metabolismo , Secuencias Repetitivas de Aminoácido , Urea
20.
Cell ; 173(3): 693-705.e22, 2018 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-29677513

RESUMEN

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.


Asunto(s)
Transporte Activo de Núcleo Celular , Señales de Localización Nuclear , Proteína FUS de Unión a ARN/química , beta Carioferinas/química , Sitios de Unión , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Carioferinas/metabolismo , Luz , Extracción Líquido-Líquido , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Mutación , Nefelometría y Turbidimetría , Unión Proteica , Dominios Proteicos , ARN/química , Dispersión de Radiación , Temperatura
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...