RESUMO
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.
Assuntos
COVID-19 , RNA Viral , Humanos , COVID-19/metabolismo , Endonucleases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação ViralRESUMO
Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , Doença de Parkinson/metabolismo , Corpos de Processamento , Estabilidade de RNA , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.
Assuntos
Interações Hospedeiro-Patógeno , RNA Viral/genética , SARS-CoV-2/genética , Animais , COVID-19/virologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Genoma Viral , Humanos , Pulmão/virologia , Masculino , Espectrometria de Massas , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/ultraestrutura , Células VeroRESUMO
The lncRNA Xist forms â¼50 diffraction-limited foci to transcriptionally silence one X chromosome. How this small number of RNA foci and interacting proteins regulate a much larger number of X-linked genes is unknown. We show that Xist foci are locally confined, contain â¼2 RNA molecules, and nucleate supramolecular complexes (SMACs) that include many copies of the critical silencing protein SPEN. Aggregation and exchange of SMAC proteins generate local protein gradients that regulate broad, proximal chromatin regions. Partitioning of numerous SPEN molecules into SMACs is mediated by their intrinsically disordered regions and essential for transcriptional repression. Polycomb deposition via SMACs induces chromatin compaction and the increase in SMACs density around genes, which propagates silencing across the X chromosome. Our findings introduce a mechanism for functional nuclear compartmentalization whereby crowding of transcriptional and architectural regulators enables the silencing of many target genes by few RNA molecules.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Longo não Codificante/metabolismo , Cromossomo X/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias , Fibroblastos , Inativação Gênica , Humanos , Camundongos , Ligação Proteica , Inativação do Cromossomo XRESUMO
Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Transcrição Gênica/genética , Fator de Transcrição YY1/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Genoma Humano/genética , Células Hep G2 , Humanos , Células K562 , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , RNA-Seq , Transcriptoma , Fator de Transcrição YY1/genéticaRESUMO
Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify â¼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Grânulos Citoplasmáticos/metabolismo , Mapas de Interação de Proteínas , Ribonucleoproteínas/metabolismo , Estresse Fisiológico , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Neurônios/metabolismo , Transporte ProteicoRESUMO
Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by â¼25% in the adult-onset cases and by â¼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.
Assuntos
Deficiências do Desenvolvimento/genética , Predisposição Genética para Doença , Haploinsuficiência/genética , Mutação/genética , Proteínas de Ligação a RNA/genética , Convulsões/genética , Adolescente , Adulto , Idade de Início , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Criança , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico por imagem , Evolução Molecular , Feminino , Deleção de Genes , Células HEK293 , Humanos , Lactente , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Neurônios/metabolismo , Neurônios/patologia , Linhagem , Estabilidade Proteica , Convulsões/diagnóstico por imagemRESUMO
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.
Assuntos
Regiões 3' não Traduzidas , Grânulos Citoplasmáticos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Fator 1 de Resposta a Butirato , Antígeno CD47/genética , Antígeno CD47/metabolismo , Grânulos Citoplasmáticos/genética , Drosophila melanogaster , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Células NIH 3T3 , Proteínas Nucleares/genética , Domínios Proteicos , Proteínas de Ligação a RNA/genéticaRESUMO
mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Mensageiro/metabolismo , Algoritmos , Sítios de Ligação , Sistema Livre de Células , Primers do DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Entropia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Dobramento de RNA , RNA Mensageiro/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ribossomos/química , Ribossomos/metabolismo , Regiões não TraduzidasRESUMO
Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.
Assuntos
Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Arginina/química , Simulação por Computador , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Transição de Fase , Proteínas Priônicas/química , Proteínas Priônicas/genética , Príons/genética , Príons/fisiologia , Domínios Proteicos , Proteína FUS de Ligação a RNA/fisiologia , Proteínas de Ligação a RNA/isolamento & purificação , Células Sf9 , Tirosina/químicaRESUMO
During eukaryotic evolution, ribosomes have considerably increased in size, forming a surface-exposed ribosomal RNA (rRNA) shell of unknown function, which may create an interface for yet uncharacterized interacting proteins. To investigate such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories including metabolism and cell cycle, as well as RNA- and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we discover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define its direct substrates. Moreover, we show that the metabolic enzyme, pyruvate kinase muscle (PKM), interacts with sub-pools of endoplasmic reticulum (ER)-associated ribosomes, exerting a non-canonical function as an RNA-binding protein in the translation of ER-destined mRNAs. Therefore, RAPs interconnect one of life's most ancient molecular machines with diverse cellular processes, providing an additional layer of regulatory potential to protein expression.
Assuntos
Ribossomos/química , Ribossomos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células-Tronco Embrionárias/metabolismo , Retículo Endoplasmático/metabolismo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Hormônios Tireóideos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.
Assuntos
Técnicas Genéticas , Príons/genética , Engenharia Genética , Técnicas Genéticas/economia , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT.
Assuntos
Neoplasias da Mama/patologia , Exossomos/patologia , RNA não Traduzido/metabolismo , Células Estromais/patologia , Microambiente Tumoral , Neoplasias da Mama/metabolismo , Proteína DEAD-box 58/metabolismo , Exossomos/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Células MCF-7 , Metástase Neoplásica , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Receptores Imunológicos , Receptores de Reconhecimento de Padrão/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Células Estromais/metabolismo , Viroses/metabolismoRESUMO
Posttranscriptional mechanisms provide powerful means to expand the coding power of genomes. In nervous systems, alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of transcripts. Thus, alternative splicing programs play instructive roles in the development of neuronal cell type-specific properties, neuronal growth, self-recognition, synapse specification, and neuronal network function. Here we discuss the most recent genome-wide efforts on mapping RNA codes and RNA-binding proteins for neuronal alternative splicing regulation. We illustrate how alternative splicing shapes key steps of neuronal development, neuronal maturation, and synaptic properties. Finally, we highlight efforts to dissect the spatiotemporal dynamics of alternative splicing and their potential contribution to neuronal plasticity and the mature nervous system.
Assuntos
Processamento Alternativo/genética , Diferenciação Celular/genética , Plasticidade Neuronal/genética , Neurônios/metabolismo , Humanos , Neurogênese/genética , Neurônios/citologia , Isoformas de Proteínas/genética , RNA/genética , Proteínas de Ligação a RNA/genética , Sinapses/genéticaRESUMO
Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.
Assuntos
Complexo Repressor Polycomb 2 , RNA Longo não Codificante , Animais , Camundongos , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromatina/genética , Sítios de LigaçãoRESUMO
The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.
Assuntos
Núcleo Celular , Splicing de RNA , Proteínas de Ligação a RNA , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Camundongos , Precursores de RNA/metabolismo , Precursores de RNA/genética , Mutação , Spliceossomos/metabolismo , Spliceossomos/genética , Células HeLa , Células HEK293RESUMO
Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.
Assuntos
Núcleo Celular , Cromatina , RNA Helicases DEAD-box , RNA Mensageiro , Animais , Humanos , Camundongos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Cromatina/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Citoplasma/genética , Estabilidade de RNA , Transporte Ativo do Núcleo Celular , Polirribossomos/metabolismo , Polirribossomos/genética , Aprendizado de Máquina , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Exossomos/metabolismo , Exossomos/genéticaRESUMO
Efficient targeted control of splicing is a major goal of functional genomics and therapeutic applications. Guide (g)RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility of these systems. However, efficient, specific, and generalizable activation of endogenous exons using this approach has not been previously reported. By screening over 300 dCasRx-splicing factor fusion proteins tethered to splicing reporters, we identify dCasRx-RBM25 as a potent activator of exons. Moreover, dCasRx-RBM25 efficiently activates the splicing of â¼90% of targeted endogenous alternative exons and displays high on-target specificity. Using gRNA arrays for combinatorial targeting, we demonstrate that dCasRx-RBM25 enables multiplexed activation and repression of exons. Using this feature, the targeting of neural-regulated exons in Ptpb1 and Puf60 in embryonic stem cells reveals combinatorial effects on downstream alternative splicing events controlled by these factors. Collectively, our results enable versatile, combinatorial exon-resolution functional assays and splicing-directed therapeutic applications.
Assuntos
Processamento Alternativo , Sistemas CRISPR-Cas , Éxons , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células HEK293 , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Animais , CamundongosRESUMO
RNA-binding proteins (RBPs) control messenger RNA fate in neurons. Here, we report a mechanism that the stimuli-induced neuronal translation is mediated by phosphorylation of a YTHDF1-binding protein FMRP. Mechanistically, YTHDF1 can condense with ribosomal proteins to promote the translation of its mRNA targets. FMRP regulates this process by sequestering YTHDF1 away from the ribosome; upon neuronal stimulation, FMRP becomes phosphorylated and releases YTHDF1 for translation upregulation. We show that a new small molecule inhibitor of YTHDF1 can reverse fragile X syndrome (FXS) developmental defects associated with FMRP deficiency in an organoid model. Our study thus reveals that FMRP and its phosphorylation are important regulators of activity-dependent translation during neuronal development and stimulation and identifies YTHDF1 as a potential therapeutic target for FXS in which developmental defects caused by FMRP depletion could be reversed through YTHDF1 inhibition.
Assuntos
Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Humanos , Fosforilação , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neurônios/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Proteínas Ribossômicas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
RNA-binding proteins (RBPs) control RNA metabolism to orchestrate gene expression and, when dysfunctional, underlie human diseases. Proteome-wide discovery efforts predict thousands of RBP candidates, many of which lack canonical RNA-binding domains (RBDs). Here, we present a hybrid ensemble RBP classifier (HydRA), which leverages information from both intermolecular protein interactions and internal protein sequence patterns to predict RNA-binding capacity with unparalleled specificity and sensitivity using support vector machines (SVMs), convolutional neural networks (CNNs), and Transformer-based protein language models. Occlusion mapping by HydRA robustly detects known RBDs and predicts hundreds of uncharacterized RNA-binding associated domains. Enhanced CLIP (eCLIP) for HydRA-predicted RBP candidates reveals transcriptome-wide RNA targets and confirms RNA-binding activity for HydRA-predicted RNA-binding associated domains. HydRA accelerates construction of a comprehensive RBP catalog and expands the diversity of RNA-binding associated domains.