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It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the fragile X-related protein 1 (FXR1) network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as an underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. Our findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as an organizer of signaling reactions.
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Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Mutação/genética , Motivos de Nucleotídeos/genética , Transição de Fase , Mutação Puntual/genética , Poli A/metabolismo , Ligação Proteica , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de SequênciaRESUMO
Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/química , Elementos Nucleotídeos Longos e Dispersos , Proteínas Associadas à Matriz Nuclear/química , Poliadenilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteínas de Ligação a RNA/química , RNA/química , Processamento Alternativo , Animais , Sítios de Ligação , Éxons , Células HeLa , Humanos , Íntrons , Camundongos , Mutação , Motivos de Nucleotídeos , Filogenia , Ligação Proteica , Mapeamento de Interação de Proteínas , Splicing de RNARESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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RNA looping adds crucial information to understanding the position-dependent regulatory mechanisms of protein-RNA interactions. In this issue, Xue et al.1 present CRIC-seq, which comprehensively identifies RNA loops mediated by specific proteins and demonstrates their value for interpreting disease-causing mutations.
Assuntos
Proteína de Ligação a Regiões Ricas em Polipirimidinas , Splicing de RNA , RNA , Processamento Alternativo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas/genética , Splicing de RNA/genéticaRESUMO
The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.
Assuntos
Retículo Endoplasmático , Proteínas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas/metabolismo , Citosol/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transporte Proteico , Biossíntese de ProteínasRESUMO
Transcription and chromatin function are regulated by proteins that bind to DNA, nucleosomes or RNA polymerase II, with specific non-coding RNAs (ncRNAs) functioning to modulate their recruitment or activity. Unlike ncRNAs, nascent pre-mRNA was considered to be primarily a passive player in these processes. In this Opinion article, we describe recently identified interactions between nascent pre-mRNAs and regulatory proteins, highlight commonalities between the functions of nascent pre-mRNA and nascent ncRNA, and propose that both types of RNA have an active role in transcription and chromatin regulation.
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Cromatina/metabolismo , Transcrição Gênica , Animais , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Splicing de RNA , Proteínas Repressoras/metabolismo , Elongação da Transcrição Genética , Fatores de TranscriçãoRESUMO
DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.
Assuntos
Linfócitos B/enzimologia , RNA Helicases DEAD-box/metabolismo , Linfoma de Células B/enzimologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-myc/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , RNA Helicases DEAD-box/genética , Estresse do Retículo Endoplasmático , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação com Perda de Função , Linfoma de Células B/genética , Linfoma de Células B/patologia , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/genética , Proteínas de Neoplasias/genética , Biossíntese de Proteínas , Proteoma , Proteostase , Proteínas Proto-Oncogênicas c-myc/genética , Adulto JovemRESUMO
There are ~650,000 Alu elements in transcribed regions of the human genome. These elements contain cryptic splice sites, so they are in constant danger of aberrant incorporation into mature transcripts. Despite posing a major threat to transcriptome integrity, little is known about the molecular mechanisms preventing their inclusion. Here, we present a mechanism for protecting the human transcriptome from the aberrant exonization of transposable elements. Quantitative iCLIP data show that the RNA-binding protein hnRNP C competes with the splicing factor U2AF65 at many genuine and cryptic splice sites. Loss of hnRNP C leads to formation of previously suppressed Alu exons, which severely disrupt transcript function. Minigene experiments explain disease-associated mutations in Alu elements that hamper hnRNP C binding. Thus, by preventing U2AF65 binding to Alu elements, hnRNP C plays a critical role as a genome-wide sentinel protecting the transcriptome. The findings have important implications for human evolution and disease.
Assuntos
Elementos Alu , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Transcriptoma , Evolução Molecular , Éxons , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Sítios de Splice de RNA , Análise de Sequência de RNA , Fator de Processamento U2AFRESUMO
RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.
Assuntos
Quinases Ciclina-Dependentes , Fosfoproteínas , Precursores de RNA , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Cromatina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Quinolonas/farmacologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/efeitos dos fármacos , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Treonina/metabolismoRESUMO
Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Proteinopatias TDP-43 , Processamento Alternativo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Códon sem Sentido , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Proteínas do Tecido Nervoso , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Due to their capacity to mediate repetitive protein interactions, intrinsically disordered regions (IDRs) are crucial for the formation of various types of protein-RNA complexes. The functions of IDRs are strongly modulated by post-translational modifications (PTMs). Phosphorylation is the most common and well-studied modification of IDRs, which can alter homomeric or heteromeric interactions of proteins and impact their ability to phase separate. Moreover, phosphorylation can influence the RNA-binding properties of proteins, and recent studies demonstrated its selective impact on the global profiles of protein-RNA binding and regulation. These findings highlight the need for further integrative approaches to understand how signalling remodels protein-RNA networks in cells.
Assuntos
Proteínas Intrinsicamente Desordenadas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA , RNA , Fosforilação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , RNA/genética , Processamento de Proteína Pós-Traducional/genética , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/químicaRESUMO
Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy remains largely unclear. Using quantitative RNA interactome capture analysis, we here reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of pancreatic cancer cells, with a network of nuclear proteins centered around nucleolin displaying enhanced RNA-binding activity. We show that nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced pancreatic cancer cell proliferation and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of nucleolin and highlight a crucial role for this mechanism in RAS-mediated tumorigenesis.
Assuntos
Genes ras , Neoplasias Pancreáticas , Humanos , Sistema de Sinalização das MAP Quinases , Proteômica , Fosfoproteínas/metabolismo , RNA Ribossômico/metabolismo , RNA/metabolismo , Transformação Celular Neoplásica/genética , Ribossomos/genética , Ribossomos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , NucleolinaRESUMO
Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.
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Venenos , Humanos , Proteínas e Peptídeos de Choque Frio/metabolismo , Temperatura Baixa , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.
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Linfócitos B/imunologia , Proteínas ELAV/imunologia , Centro Germinativo/imunologia , Imunidade Humoral , Imunoglobulinas/biossíntese , RNA Mensageiro/imunologia , Aciltransferases/genética , Aciltransferases/imunologia , Processamento Alternativo/imunologia , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Morte Celular , Diferenciação Celular , Proliferação de Células , Proteínas ELAV/genética , Eritrócitos/imunologia , Centro Germinativo/citologia , Centro Germinativo/efeitos dos fármacos , Imunização , Switching de Imunoglobulina , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/imunologia , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , OvinosRESUMO
The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.
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Biossíntese de Proteínas/genética , Proteostase/genética , RNA Antissenso/genética , Tauopatias/genética , Tauopatias/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Idoso , Animais , Sítios de Ligação , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Diferenciação Celular , Progressão da Doença , Feminino , Humanos , Sítios Internos de Entrada Ribossomal/genética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Ribossomos/metabolismo , Proteínas tau/biossínteseRESUMO
High-throughput sequencing-based methods and their applications in the study of transcriptomes have revolutionized our understanding of alternative splicing. Networks of functionally coordinated and biologically important alternative splicing events continue to be discovered in an ever-increasing diversity of cell types in the context of physiologically normal and disease states. These studies have been complemented by efforts directed at defining sequence codes governing splicing and their cognate trans-acting factors, which have illuminated important combinatorial principles of regulation. Additional studies have revealed critical roles of position-dependent, multivalent protein-RNA interactions that direct splicing outcomes. Investigations of evolutionary changes in RNA binding proteins, splice variants, and associated cis elements have further shed light on the emergence, mechanisms, and functions of splicing networks. Progress in these areas has emphasized the need for a coordinated, community-based effort to systematically address the functions of individual splice variants associated with normal and disease biology.
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Processamento Alternativo/fisiologia , Evolução Molecular , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , HumanosRESUMO
DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.
Assuntos
Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Apoptose/genética , Sobrevivência Celular/genética , Dano ao DNA/genética , Células HEK293 , Humanos , RNA Longo não Codificante/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.
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Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias Murinas/metabolismo , RNA Longo não Codificante/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Células-Tronco Pluripotentes/metabolismo , Poliadenilação/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
RNA binding proteins (RBPs) regulate all aspects in the life cycle of RNA molecules. To elucidate the elements that guide RNA specificity, regulatory mechanisms, and functions of RBPs, methods that identify direct endogenous protein-RNA interactions are particularly valuable. UV crosslinking and immunoprecipitation (CLIP) purifies short RNA fragments that crosslink to a specific protein and then identifies these fragments by sequencing. When combined with high-throughput sequencing, CLIP can produce transcriptome-wide maps of RNA crosslink sites. The protocol is comprised of several dozen biochemical steps, and improvements made over the last 15 years have increased its resolution, sensitivity, and convenience. Adaptations of CLIP are also emerging in the epitranscriptomic field to map the positions of RNA modifications accurately. Here, we describe the rationale for each step in the protocol and discuss the impact of variations to help users determine the most suitable option.