RESUMEN
The engagement of a T cell with an antigen-presenting cell (APC) or activating surface results in the formation within the T cell of several distinct actin and actomyosin networks. These networks reside largely within a narrow zone immediately under the T cell's plasma membrane at its site of contact with the APC or activating surface, i.e., at the immunological synapse. Here we review the origin, organization, dynamics, and function of these synapse-associated actin and actomyosin networks. Importantly, recent insights into the nature of these actin-based cytoskeletal structures were made possible in several cases by advances in light microscopy.
Asunto(s)
Actinas/metabolismo , Actomiosina/metabolismo , Células Presentadoras de Antígenos/metabolismo , Citoesqueleto/metabolismo , Sinapsis Inmunológicas/metabolismo , Linfocitos T/metabolismo , Animales , Presentación de Antígeno , Humanos , Activación de LinfocitosRESUMEN
Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.
Asunto(s)
Envejecimiento , Encéfalo , Complemento C1q , Homeostasis , Microglía , Neuronas , Ribonucleoproteínas , Animales , Complemento C1q/metabolismo , Ratones , Microglía/metabolismo , Envejecimiento/metabolismo , Encéfalo/metabolismo , Ribonucleoproteínas/metabolismo , Neuronas/metabolismo , Ratones Endogámicos C57BL , HumanosRESUMEN
Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Animales , Ratones , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Citoplasma , Neuronas Motoras , Proteínas con Motivos de Reconocimiento de ARN/metabolismoRESUMEN
N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.
Asunto(s)
ADN Helicasas , ARN Helicasas , ADN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , ARN Helicasas/metabolismo , Gránulos de Estrés , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión al GTP/metabolismo , ARN Mensajero/metabolismo , Gránulos Citoplasmáticos/metabolismoRESUMEN
Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.
Asunto(s)
Condensados Biomoleculares/metabolismo , Gránulos Citoplasmáticos/metabolismo , Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación Enzimática , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Células HEK293 , Humanos , Proteína SOS1/metabolismo , Transducción de SeñalRESUMEN
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Helicasas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Esclerosis Tuberosa/metabolismo , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/química , Evolución Molecular , Femenino , Humanos , Insulina/farmacología , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa/química , ARN Helicasas/química , Proteínas con Motivos de Reconocimiento de ARN/química , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Pez Cebra/metabolismoRESUMEN
The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Estructuras Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilación , ARN/metabolismoRESUMEN
Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, and to better understand how RNA-based condensation occurs, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates in vitro. We demonstrate that, through ATP-dependent RNA binding, the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a function for eIF4A to limit intermolecular RNA-RNA interactions in cells. These results establish an important role for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , ARN Helicasas/metabolismo , ARN de Hongos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Unión Proteica , ARN de Hongos/aislamiento & purificación , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Imagen de Lapso de TiempoRESUMEN
Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Bacterias/inmunología , Movimiento Celular/inmunología , Células Asesinas Naturales/inmunología , Trofoblastos/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Células Dendríticas/inmunología , Femenino , Células HeLa , Humanos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Placenta/inmunología , Placenta/microbiología , Embarazo , Ratas , Células THP-1 , Trofoblastos/microbiologíaRESUMEN
Throughout mammalian neocortex, layer 5 pyramidal (L5) cells project via the pons to a vast number of cerebellar granule cells (GrCs), forming a fundamental pathway. Yet, it is unknown how neuronal dynamics are transformed through the L5âGrC pathway. Here, by directly comparing premotor L5 and GrC activity during a forelimb movement task using dual-site two-photon Ca2+ imaging, we found that in expert mice, L5 and GrC dynamics were highly similar. L5 cells and GrCs shared a common set of task-encoding activity patterns, possessed similar diversity of responses, and exhibited high correlations comparable to local correlations among L5 cells. Chronic imaging revealed that these dynamics co-emerged in cortex and cerebellum over learning: as behavioral performance improved, initially dissimilar L5 cells and GrCs converged onto a shared, low-dimensional, task-encoding set of neural activity patterns. Thus, a key function of cortico-cerebellar communication is the propagation of shared dynamics that emerge during learning.
Asunto(s)
Cerebelo/metabolismo , Neocórtex/metabolismo , Animales , Conducta Animal , Calcio/metabolismo , Miembro Anterior/fisiología , Ratones , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica , Neocórtex/patología , Opsinas/genética , Opsinas/metabolismo , Células Piramidales/metabolismoRESUMEN
Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.
Asunto(s)
Anexinas/metabolismo , Transporte Axonal/fisiología , Gránulos Citoplasmáticos/metabolismo , Lisosomas/metabolismo , ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Anexinas/genética , Axones/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mutación , Unión Proteica , Ratas/embriología , Ratas Sprague-Dawley , Transfección , Pez CebraRESUMEN
Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral/patología , Ataxina-2/metabolismo , Proteína C9orf72/genética , Demencia Frontotemporal/patología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Arsenitos/toxicidad , Ataxina-2/antagonistas & inhibidores , Ataxina-2/genética , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN/genética , Femenino , Demencia Frontotemporal/metabolismo , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Proteínas de Complejo Poro Nuclear/metabolismo , Estrés Oxidativo/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Compuestos de Sodio/toxicidad , alfa Carioferinas/antagonistas & inhibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismoRESUMEN
Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.
Asunto(s)
Arginina/química , Chaperonas Moleculares/química , Proteína FUS de Unión a ARN/química , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Cationes , Metilación de ADN , Demencia Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Unión Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteína FUS de Unión a ARN/metabolismo , Tirosina/química , Xenopus laevisRESUMEN
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 , TemperaturaRESUMEN
Approximately half of human genes generate mRNAs with alternative 3' untranslated regions (3'UTRs). Through 3'UTR-mediated protein-protein interactions, alternative 3'UTRs enable multi-functionality of proteins with identical amino acid sequence. While studying how information on protein features is transferred from 3'UTRs to proteins, we discovered that the broadly expressed RNA-binding protein TIS11B forms a membraneless organelle, called TIS granule, that enriches membrane protein-encoding mRNAs with multiple AU-rich elements. TIS granules form a reticular meshwork intertwined with the endoplasmic reticulum (ER). The association between TIS granules and the ER creates a subcellular compartment-the TIGER domain-with a biophysically and biochemically distinct environment from the cytoplasm. This compartment promotes 3'UTR-mediated interaction of SET with membrane proteins, thus allowing increased surface expression and functional diversity of proteins, including CD47 and PD-L1. The TIGER domain is a subcellular compartment that enables formation of specific and functionally relevant protein-protein interactions that cannot be established outside.
Asunto(s)
Regiones no Traducidas 3' , Gránulos Citoplasmáticos/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Factor 1 de Respuesta al Butirato , Antígeno CD47/genética , Antígeno CD47/metabolismo , Gránulos Citoplasmáticos/genética , Drosophila melanogaster , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Células MCF-7 , Ratones , Células 3T3 NIH , Proteínas Nucleares/genética , Dominios Proteicos , Proteínas de Unión al ARN/genéticaRESUMEN
Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly.
Asunto(s)
Arabidopsis , Respuesta al Choque Térmico , Complejo de la Endopetidasa Proteasomal , Ubiquitinación , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteolisis , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Gránulos Citoplasmáticos/metabolismoRESUMEN
mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
Asunto(s)
ARN Mensajero , Proteínas de Unión al ARN , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Unión Proteica , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Células HeLa , Factores de Tiempo , Aprendizaje AutomáticoRESUMEN
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Dipéptidos/metabolismo , Demencia Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteína C9orf72 , Nucléolo Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Expansión de las Repeticiones de ADN , Dipéptidos/genética , Drosophila melanogaster/genética , Demencia Frontotemporal/genética , Humanos , Membranas Intracelulares/metabolismo , Poro Nuclear/metabolismo , Péptidos/genética , Péptidos/metabolismo , Proteínas/genéticaRESUMEN
Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the RIG-I-like receptors (RLRs), which recognize viral dsRNA and initiate antiviral innate immune responses with the potential of triggering systemic inflammation and immunopathology. Here, we show that stress granules (SGs), molecular condensates that form in response to various stresses including viral dsRNA, play key roles in the controlled activation of RLR signaling. Without the SG nucleators G3BP1/2 and UBAP2L, dsRNA triggers excessive inflammation and immune-mediated apoptosis. In addition to exogenous dsRNA, host-derived dsRNA generated in response to ADAR1 deficiency is also controlled by SG biology. Intriguingly, SGs can function beyond immune control by suppressing viral replication independently of the RLR pathway. These observations thus highlight the multi-functional nature of SGs as cellular "shock absorbers" that converge on protecting cell homeostasis by dampening both toxic immune response and viral replication.
Asunto(s)
ADN Helicasas , ARN Helicasas , Humanos , ADN Helicasas/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Gránulos de Estrés , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Inmunidad Innata , Inflamación/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.