RESUMEN
Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
Asunto(s)
COVID-19 , ARN Viral , Humanos , COVID-19/metabolismo , Endonucleasas/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/genética , Replicación ViralRESUMEN
Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.
Asunto(s)
Proteína de Replicación A , Expansión de Repetición de Trinucleótido , Animales , Humanos , Ratones , ADN/genética , Reparación de la Incompatibilidad de ADN , Enfermedad de Huntington/genética , Proteínas/genética , Ataxias Espinocerebelosas/genética , Proteína de Replicación A/metabolismoRESUMEN
Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.
Asunto(s)
Mitocondrias/metabolismo , Degeneración Nerviosa/metabolismo , Mapas de Interacción de Proteínas , Sinapsis/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Aminoácidos/metabolismo , Biotinilación , Encéfalo/metabolismo , Encéfalo/patología , Núcleo Celular/metabolismo , Progresión de la Enfermedad , Metabolismo Energético , Demencia Frontotemporal/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Degeneración Nerviosa/patología , Neuronas/metabolismo , Unión Proteica , Dominios Proteicos , Proteómica , Índice de Severidad de la Enfermedad , Fracciones Subcelulares/metabolismo , Tauopatías/genética , Proteínas tau/químicaRESUMEN
Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.
Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas B-raf , Carcinogénesis , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Mutación , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.
Asunto(s)
Factor de Transcripción GATA4/metabolismo , Cardiopatías Congénitas , Proteínas Nucleares/metabolismo , Oxidorreductasas/metabolismo , Factores de Transcripción , Animales , Cardiopatías Congénitas/genética , Ratones , Mutación , Proteómica , Proteínas de Dominio T Box/genética , Factores de Transcripción/genéticaRESUMEN
Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Proteoma/genética , Biología Computacional/métodos , Células HCT116/metabolismo , Células HEK293/metabolismo , Humanos , Espectrometría de Masas/métodos , Mapas de Interacción de Proteínas/fisiología , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed â¼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.
Asunto(s)
Ligandos , Mapas de Interacción de Proteínas/fisiología , Receptores de Superficie Celular/metabolismo , Receptor DCC/química , Receptor DCC/metabolismo , Humanos , Filogenia , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/clasificación , Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/química , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Cell surface receptors and their interactions play a central role in physiological and pathological signaling. Despite its clinical relevance, the immunoglobulin superfamily (IgSF) remains uncharacterized and underrepresented in databases. Here, we present a systematic extracellular protein map, the IgSF interactome. Using a high-throughput technology to interrogate most single transmembrane receptors for binding to 445 IgSF proteins, we identify over 500 interactions, 82% previously undocumented, and confirm more than 60 receptor-ligand pairs using orthogonal assays. Our study reveals a map of cell-type-specific interactions and the landscape of dysregulated receptor-ligand crosstalk in cancer, including selective loss of function for tumor-associated mutations. Furthermore, investigation of the IgSF interactome in a large cohort of cancer patients identifies interacting protein signatures associated with clinical outcome. The IgSF interactome represents an important resource to fuel biological discoveries and a framework for understanding the functional organization of the surfaceome during homeostasis and disease, ultimately informing therapeutic development.
Asunto(s)
Inmunoglobulinas/metabolismo , Neoplasias/patología , Mapas de Interacción de Proteínas , Antígeno B7-H1/metabolismo , Antígeno Carcinoembrionario/metabolismo , Comunicación Celular , Análisis por Conglomerados , Medios de Cultivo Condicionados/química , Células HEK293 , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/genética , Ligandos , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.
Asunto(s)
Mapeo Cromosómico/métodos , Trastornos del Neurodesarrollo/genética , Biología de Sistemas/métodos , Redes Reguladoras de Genes/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos , Humanos , Neurobiología/métodos , NeuropsiquiatríaRESUMEN
Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells, but our knowledge of the metabolite-protein interactome lags behind our understanding of protein-protein or protein-DNA interactomes. Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environment. The approach identified a network of known and novel interactions and binding sites in Escherichia coli, and we demonstrated the functional relevance of a number of newly identified interactions. Our data enabled identification of new enzyme-substrate relationships and cases of metabolite-induced remodeling of protein complexes. Our metabolite-protein interactome consists of 1,678 interactions and 7,345 putative binding sites. Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.
Asunto(s)
Metaboloma , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal , Programas Informáticos , Regulación Alostérica , Sitios de Unión , Escherichia coli , Metabolómica/métodos , Unión Proteica , Mapas de Interacción de Proteínas , Proteoma/química , Saccharomyces cerevisiae , Análisis de Secuencia de Proteína/métodosRESUMEN
To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR â Gαo â Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.
Asunto(s)
Membrana Celular/metabolismo , Extensiones de la Superficie Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Aparato de Golgi/metabolismo , Animales , Línea Celular , Drosophila , Femenino , GTP Fosfohidrolasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.
RESUMEN
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
Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated.
Asunto(s)
Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Regulación de la Expresión Génica , Insulina/genética , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Retroalimentación Fisiológica , Humanos , Insulina/metabolismo , Membranas Intracelulares/metabolismo , Fosforilación , Transporte de Proteínas , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Mitochondrial sirtuins, SIRT3-5, are NAD+-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.
Asunto(s)
Mitocondrias/metabolismo , Mapas de Interacción de Proteínas , Sirtuina 3/metabolismo , Acetilación , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales , Sirtuinas/clasificación , Sirtuinas/metabolismoRESUMEN
All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.
Asunto(s)
Péptidos , Biosíntesis de Proteínas , Humanos , Sistemas de Lectura Abierta , Péptidos/genética , Proteómica , MicropéptidosRESUMEN
Epstein-Barr virus (EBV) causes infectious mononucleosis, triggers multiple sclerosis, and is associated with 200,000 cancers/year. EBV colonizes the human B cell compartment and periodically reactivates, inducing expression of 80 viral proteins. However, much remains unknown about how EBV remodels host cells and dismantles key antiviral responses. We therefore created a map of EBV-host and EBV-EBV interactions in B cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. The EBV-encoded G-protein-coupled receptor BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Although UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication activated the NLRP3 inflammasome, which impaired viral replication and triggered pyroptosis. Our results provide a viral protein interaction network resource, reveal a UFM1-dependent pathway for selective degradation of mitochondrial cargo, and highlight BILF1 as a novel therapeutic target.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/genética , Infecciones por Virus de Epstein-Barr/genética , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Mapas de Interacción de ProteínasRESUMEN
Cellular mRNA-binding proteins (mRBPs) are major posttranscriptional regulators of gene expression. Although many posttranslational modification sites in mRBPs have been identified, little is known about how these modifications regulate mRBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and nonphosphorylated forms of mRBPs. Almost 200 phosphorylation events affected pull-down efficiency compared with the unmodified mRBPs and thus have regulatory potential. Our data capture known regulatory phosphorylation sites in ELAVL1, SF3B1, and UPF1 and identify potential regulatory sites. Follow-up experiments on the splicing regulator RBM20 revealed multiple phosphorylation sites in the C-terminal disordered region affecting nucleocytoplasmic localization, association with cytoplasmic ribonucleoprotein granules, and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in mRBPs.
Asunto(s)
Proteoma , Proteínas de Unión al ARN , Fosforilación , Proteoma/genética , Proteoma/metabolismo , ARN/metabolismo , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRNPs) are rearranged and undergo quality control prior to export, ensuring that only mature mRNAs reach the cytoplasm. Here, we use proteomic, genetic, live-cell, and single-molecule resolution microscopy approaches in budding yeast to demonstrate that basket formation is dependent on RNA polymerase II transcription and subsequent mRNP processing. We further show that while all NPCs can bind Mlp1, baskets assemble only on a subset of nucleoplasmic NPCs, and these basket-containing NPCs associate a distinct protein and RNA interactome. Taken together, our data point toward NPC heterogeneity and an RNA-dependent mechanism for functionalization of NPCs in budding yeast through nuclear basket assembly.
Asunto(s)
Poro Nuclear , Saccharomycetales , Poro Nuclear/genética , Poro Nuclear/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteómica , Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.