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1.
Cell ; 184(18): 4680-4696.e22, 2021 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-34380047

RESUMEN

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Mutación/genética , Motivos de Nucleótidos/genética , Transición de Fase , Mutación Puntual/genética , Poli A/metabolismo , Unión Proteica , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de Secuencia
2.
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
3.
Mol Cell ; 69(3): 465-479.e7, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29358076

RESUMEN

hnRNPA2, a component of RNA-processing membraneless organelles, forms inclusions when mutated in a syndrome characterized by the degeneration of neurons (bearing features of amyotrophic lateral sclerosis [ALS] and frontotemporal dementia), muscle, and bone. Here we provide a unified structural view of hnRNPA2 self-assembly, aggregation, and interaction and the distinct effects of small chemical changes-disease mutations and arginine methylation-on these assemblies. The hnRNPA2 low-complexity (LC) domain is compact and intrinsically disordered as a monomer, retaining predominant disorder in a liquid-liquid phase-separated form. Disease mutations D290V and P298L induce aggregation by enhancing and extending, respectively, the aggregation-prone region. Co-aggregating in disease inclusions, hnRNPA2 LC directly interacts with and induces phase separation of TDP-43. Conversely, arginine methylation reduces hnRNPA2 phase separation, disrupting arginine-mediated contacts. These results highlight the mechanistic role of specific LC domain interactions and modifications conserved across many hnRNP family members but altered by aggregation-causing pathological mutations.


Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Arginina/genética , Arginina/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Imagen por Resonancia Magnética/métodos , Metilación , Mutación , Neuronas/metabolismo , Neuronas/patología , Procesamiento Proteico-Postraduccional
4.
Proc Natl Acad Sci U S A ; 120(34): e2305625120, 2023 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-37579155

RESUMEN

TAR DNA-binding protein 43 (TDP-43) is involved in key processes in RNA metabolism and is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone, can undergo liquid-liquid phase separation (LLPS) in isolation, and is critical for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (QN-rich, IDR1/2) are also found to play a critical role in PS and aggregation. Compared with other phase-separating proteins, TDP-43 CTD has a notably distinct sequence composition including many aliphatic residues such as methionine and leucine. Aliphatic residues were previously suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution toward CTD phase separation has been relatively ignored. Using multiscale simulations coupled with in vitro saturation concentration (csat) measurements, we identified the importance of aromatic residues while also suggesting an essential role for aliphatic methionine residues in promoting single-chain compaction and LLPS. Surprisingly, NMR experiments showed that transient interactions involving phenylalanine and methionine residues in the disordered flanking regions can directly enhance site-specific, CR-mediated intermolecular association. Overall, our work highlights an underappreciated mode of biomolecular recognition, wherein both transient and site-specific hydrophobic interactions act synergistically to drive the oligomerization and phase separation of a disordered, low-complexity domain.


Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Dominios Proteicos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/genética , Proteínas de Unión al ADN/metabolismo , Metionina
5.
EMBO J ; 40(3): e105001, 2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33349959

RESUMEN

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch-TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress-induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp-1, suggesting gain-of-function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.


Asunto(s)
Caenorhabditis elegans/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Enfermedades Neurodegenerativas/genética , Tirosina/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Modelos Animales de Enfermedad , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Modelos Moleculares , Degeneración Nerviosa , Enfermedades Neurodegenerativas/metabolismo , Fosforilación , Conformación Proteica , Dominios Proteicos
6.
Mol Cell ; 65(6): 1044-1055.e5, 2017 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-28306503

RESUMEN

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 , Transfección
7.
Biophys J ; 123(11): 1481-1493, 2024 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-38297837

RESUMEN

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Efg1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered but have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase-separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans transcription factor interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.


Asunto(s)
Proteínas Fúngicas , Dominios Proteicos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Candida albicans/metabolismo , Priones/metabolismo , Priones/química , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Separación de Fases , Proteínas de Unión al ADN
8.
EMBO J ; 39(24): e106478, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33200826

RESUMEN

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA-binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) condenses with RNA via liquid-liquid phase separation (LLPS) and that N protein can be recruited in phase-separated forms of human RNA-binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N-terminal IDR and central-linker IDR, as well as the folded C-terminal oligomerization domain, while the folded N-terminal domain and the C-terminal IDR are not required. N protein phase separation is induced by addition of non-specific RNA. In addition, N partitions in vitro into phase-separated forms of full-length human hnRNPs (TDP-43, FUS, hnRNPA2) and their low-complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS-CoV-2 viral genome packing and in host-protein co-opting necessary for viral replication and infectivity.


Asunto(s)
COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/química , SARS-CoV-2/química , COVID-19/patología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Gránulos Citoplasmáticos/virología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Interacciones Huésped-Patógeno , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/metabolismo , SARS-CoV-2/metabolismo , Ensamble de Virus
9.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33688043

RESUMEN

Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes.


Asunto(s)
Membrana Celular/química , Fuerza Compresiva , Proteínas de la Membrana/química , Estrés Mecánico , Humanos , Conformación Proteica
10.
J Cell Sci ; 134(17)2021 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-34357401

RESUMEN

Myxoid liposarcoma is caused by a chromosomal translocation resulting in a fusion protein comprised of the N terminus of FUS (fused in sarcoma) and the full-length transcription factor CHOP (CCAAT/enhancer-binding protein homologous protein, also known as DDIT3). FUS functions in RNA metabolism, and CHOP is a stress-induced transcription factor. The FUS-CHOP fusion protein causes unique gene expression and oncogenic transformation. Although it is clear that the FUS segment is required for oncogenic transformation, the mechanism of FUS-CHOP-induced transcriptional activation is unknown. Recently, some transcription factors and super enhancers have been proposed to undergo liquid-liquid phase separation and form membraneless compartments that recruit transcription machinery to gene promoters. Since phase separation of FUS depends on its N terminus, transcriptional activation by FUS-CHOP could result from the N terminus driving nuclear phase transitions. Here, we characterized FUS-CHOP in cells and in vitro, and observed novel phase-separating properties relative to unmodified CHOP. Our data indicate that FUS-CHOP forms phase-separated condensates that colocalize with BRD4, a marker of super enhancer condensates. We provide evidence that the FUS-CHOP phase transition is a novel oncogenic mechanism and potential therapeutic target for myxoid liposarcoma. This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas de Ciclo Celular , Humanos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína FUS de Unión a ARN/genética , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética
11.
Mol Cell ; 60(2): 231-41, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26455390

RESUMEN

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.


Asunto(s)
Gránulos Citoplasmáticos/química , Proteínas Intrínsecamente Desordenadas/química , ARN Polimerasa II/química , Proteína FUS de Unión a ARN/química , Proteínas de Unión al ARN/química , ARN/química , Secuencias de Aminoácidos , Sitios de Unión , Gránulos Citoplasmáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Imitación Molecular , Datos de Secuencia Molecular , Transición de Fase , Priones/química , Priones/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reología
12.
Proc Natl Acad Sci U S A ; 117(11): 5883-5894, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32132204

RESUMEN

Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Conformación Proteica en Hélice alfa , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Humanos , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Mutación , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Empalme de Proteína , Proteínas de Unión al ARN/metabolismo
13.
EMBO J ; 37(5)2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29438978

RESUMEN

TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS) in vitro, fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation and in vitro biochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Agregación Patológica de Proteínas/genética , Dominios Proteicos/genética , Empalme del ARN/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Polimerizacion , Polímeros/metabolismo , Agregación Patológica de Proteínas/patología
14.
EMBO J ; 37(11)2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29764981

RESUMEN

TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Proteínas de Unión al ARN/genética , Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Exones/genética , Humanos , Ratones , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mutación , Empalme del ARN/genética
15.
Nucleic Acids Res ; 48(18): 10542-10554, 2020 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-32870271

RESUMEN

hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts.


Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Motivo de Reconocimiento de ARN/genética , Proteínas de Unión al ARN/genética , Elementos de Respuesta/genética , Sitios de Unión/genética , Fenómenos Biofísicos , Humanos , Extracción Líquido-Líquido , Espectroscopía de Resonancia Magnética , Neuronas/metabolismo , Oligodendroglía/metabolismo , Agregado de Proteínas/genética , Unión Proteica/genética , ARN/genética
16.
Biophys J ; 120(7): 1187-1197, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33582133

RESUMEN

Biomolecules undergo liquid-liquid phase separation (LLPS), resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post-translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed-charge atomistic force fields, we parameterize the size and atomistic hydropathy of the coarse-grained-modified amino acid beads and, hence, the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein's single-chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of Fused in Sarcoma (FUS) and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution.


Asunto(s)
Proteínas Intrínsecamente Desordenadas , Simulación por Computador , Proteínas Intrínsecamente Desordenadas/metabolismo , Orgánulos/metabolismo , Procesamiento Proteico-Postraduccional
17.
Biophys J ; 120(11): 2181-2191, 2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-33798566

RESUMEN

Long interspersed nuclear element-1 (L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as messenger RNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation, whereas some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Chaperonas Moleculares , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Sistemas de Lectura Abierta , Dominios Proteicos , ARN Mensajero
18.
J Biol Chem ; 295(8): 2375-2384, 2020 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-31911439

RESUMEN

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a phenomenon that underlies membraneless compartmentalization of the cell. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membraneless organelles in facilitating various biological processes and the disease association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions and remain dynamic in solution. Solution-state NMR spectroscopy has emerged as a leading structural technique to characterize protein LLPS due to the variety and specificity of information that can be obtained about intrinsically disordered sequences. This review discusses practical aspects of studying LLPS by NMR, summarizes recent work on the molecular aspects of LLPS of various protein systems, and discusses future opportunities for characterizing the molecular details of LLPS to modulate phase separation.


Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Espectroscopía de Resonancia Magnética , Movimiento (Física) , Conformación Proteica
19.
EMBO J ; 36(20): 2951-2967, 2017 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-28790177

RESUMEN

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/química
20.
J Biol Chem ; 293(51): 19522-19531, 2018 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-30397184

RESUMEN

Liquid-liquid phase separation of proteins and nucleic acids into membraneless organelles (MLOs) spatially organizes cellular components and reactions. The RNA-binding protein heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) carries mRNA targets in MLOs called transport granules in neurons and oligodendrocytes. At sites of local translation, hnRNPA2 is phosphorylated by the tyrosine protein kinase Fyn, releasing the mRNA for translation. Fyn recognizes targets through its SH3 domain (Fyn-SH3). However, hnRNPA2 lacks canonical SH3-binding sequences, raising the question of how Fyn-SH3 binds hnRNPA2 in phase-separated transport granules. Here, we characterize the structural details of the interaction of the hnRNPA2 low-complexity domain (LC) with Fyn-SH3 and the effect of Fyn-SH3 on hnRNPA2 phase separation. We combined in vitro microscopy and solution NMR spectroscopy to evaluate assembly of hnRNPA2 and Fyn-SH3 into in vitro phase-separated granules and probe the structural details of their interaction. We observed that Fyn-SH3 induces hnRNPA2 LC phase separation and that Fyn-SH3 is incorporated into in vitro hnRNPA2 LC granules. Moreover, we identified hnRNPA2 LC interaction sites on the surface of Fyn-SH3. Our data offer a structural view of how hnRNPA2 LC may interact with Fyn. To our knowledge, our study provides the first example of a single globular domain inducing phase separation of a disordered MLO scaffold protein.


Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Proteínas Proto-Oncogénicas c-fyn/química , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Dominios Homologos src , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Modelos Moleculares , Mutación , Unión Proteica
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