RESUMO
The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs). Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3' untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions. We also discover a duplex spanning 858 nucleotides in the 3' UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-BoxRESUMO
Cytoplasmic polyadenylation-induced translation controls germ cell development, neuronal synaptic plasticity and cellular senescence, a tumour-suppressor mechanism that limits the replicative lifespan of cells. The cytoplasmic polyadenylation element binding protein (CPEB) promotes polyadenylation by nucleating a group of factors including defective in germline development 2 (Gld2), a non-canonical poly(A) polymerase, on specific messenger RNA (mRNA) 3' untranslated regions (UTRs). Because CPEB regulation of p53 mRNA polyadenylation/translation is necessary for cellular senescence in primary human diploid fibroblasts, we surmised that Gld2 would be the enzyme responsible for poly(A) addition. Here we show that depletion of Gld2 surprisingly promotes rather than inhibits p53 mRNA polyadenylation/translation, induces premature senescence and enhances the stability of CPEB mRNA. The CPEB 3' UTR contains two miR-122 binding sites, which when deleted, elevate mRNA translation, as does an antagomir of miR-122. Although miR-122 is thought to be liver specific, it is present in primary fibroblasts and destabilized by Gld2 depletion. Gld4, a second non-canonical poly(A) polymerase, was found to regulate p53 mRNA polyadenylation/translation in a CPEB-dependent manner. Thus, translational regulation of p53 mRNA and cellular senescence is coordinated by Gld2/miR-122/CPEB/Gld4.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Poliadenilação , Polinucleotídeo Adenililtransferase/genéticaRESUMO
TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3' UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3' UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Sequência de Bases , Northern Blotting , Linhagem Celular , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Humanos , Immunoblotting , Imunoprecipitação , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase , Interferência de RNA , Análise de Sequência de DNARESUMO
RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.
Assuntos
Biblioteca Gênica , Imunoprecipitação/métodos , Proteínas de Ligação a RNA/química , RNA/química , Sítios de Ligação , DNA Circular/química , DNA Circular/genética , Regulação da Expressão Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , RNA/genética , Proteínas de Ligação a RNA/genética , Raios UltravioletaRESUMO
Nuclear factor TDP-43 has been reported to play multiple roles in transcription, pre-mRNA splicing, mRNA stability and mRNA transport. From a structural point of view, TDP-43 is a member of the hnRNP protein family whose structure includes two RRM domains flanked by the N-terminus and C-terminal regions. Like many members of this family, the C-terminal region can interact with cellular factors and thus serve to modulate its function. Previously, we have described that TDP-43 binds to several members of the hnRNP A/B family through this region. In this work, we set up a coupled minigene/siRNA cellular system that allows us to obtain in vivo data to address the functional significance of TDP-43-recruited hnRNP complex formation. Using this method, we have finely mapped the interaction between TDP-43 and the hnRNP A2 protein to the region comprised between amino acid residues 321 and 366. Our results provide novel details of protein-protein interactions in splicing regulation. In addition, we provide further insight on TDP-43 functional properties, particularly the lack of effects, as seen with our assays, of the disease-associated mutations that fall within the TDP-43 321-366 region: Q331K, M337V and G348C.
Assuntos
Proteínas de Ligação a DNA/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Splicing de RNA , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Peptídeos/farmacologia , Mapeamento de Interação de Proteínas , Interferência de RNA , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
TAR DNA binding protein (TDP43), a highly conserved heterogeneous nuclear ribonucleoprotein, was found to down-regulate splicing of the exon 9 cystic fibrosis transmembrane conductance regulator (CFTR) through specific binding to a UG-rich polymorphic region upstream of the 3' splice site. Despite the emergence of new information regarding the protein's nuclear localization and splicing regulatory activity, TDP43's role in cells remains elusive. To investigate the function of human TDP43 and its homologues, we cloned and characterized the proteins from Drosophila melanogaster and Caenorhabditis elegans. The proteins from human, fly, and worm show striking similarities in their nucleic acid binding specificity. We found that residues at two different positions, which show a strong conservation among TDP43 family members, are linked to the tight recognition of the target sequence. Our three-dimensional model of TDP43 in complex with a (UG)(m) sequence predicts that these residues make amino acid side-chain to base contacts. Moreover, our results suggest that Drosophila TDP43 is comparable to human TDP43 in regulating exon splicing. On the other hand, C.elegans TDP43 has no effect on exon recognition. TDP43 from C.elegans lacks the glycine-rich domain found at the carboxy terminus of the other two homologues. Mutants of human and fly TDP43 devoid of the C-terminal domain are likewise unable to affect splicing. Our studies suggest that the glycine-rich domain is essential for splicing regulation by human and fly TDP43.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/metabolismo , Ácidos Nucleicos/metabolismo , Splicing de RNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
The cytoplasmic polyadenylation element binding proteins (CPEBs) associate with specific sequences in mRNA 3' untranslated regions to promote translation. They do so by inducing cytoplasmic polyadenylation, which requires specialized poly(A) polymerases. Aberrant expression of these proteins correlates with certain types of cancer, indicating that cytoplasmic RNA 3' end processing is important in the control of growth. Several CPEB-regulated mRNAs govern cell cycle progression, regulate senescence, establish cell polarity, and promote tumorigenesis and metastasis. In this Opinion article, we discuss the emerging evidence that indicates a key role for the CPEBs in cancer biology.
Assuntos
Neoplasias , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/fisiologia , Fatores de Poliadenilação e Clivagem de mRNA/fisiologia , Animais , Ciclo Celular/fisiologia , Proliferação de Células , Senescência Celular/fisiologia , Humanos , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Polinucleotídeo Adenililtransferase , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The noncanonical polymerase Gld2 has been implicated in the stabilization of miR-122, possibly through catalyzing 3' monoadenylation; however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2's involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3' end. These results establish a mechanism of miRNA stability and resulting posttranscriptional gene regulation.
Assuntos
Fibroblastos/metabolismo , MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Estabilidade de RNA/fisiologia , Animais , Linhagem Celular , Fibroblastos/citologia , Humanos , Camundongos , MicroRNAs/genética , Polinucleotídeo Adenililtransferase/genética , Fatores de Poliadenilação e Clivagem de mRNARESUMO
Pathological modifications in the highly conserved and ubiquitously expressed heterogeneous ribonucleoprotein TDP-43 were recently associated to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a late-onset disorder that affects predominantly motoneurons [Neumann, M. et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130-133, Sreedharan, J. et al. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668-1672, Kabashi, E. et al. (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat. Genet. 40, 572-574]. However, the function of TDP-43 in vivo is unknown and a possible direct role in neurodegeneration remains speculative. Here, we report that flies lacking Drosophila TDP-43 appeared externally normal but presented deficient locomotive behaviors, reduced life span and anatomical defects at the neuromuscular junctions. These phenotypes were rescued by expression of the human protein in a restricted group of neurons including motoneurons. Our results demonstrate the role of this protein in vivo and suggest an alternative explanation to ALS pathogenesis that may be more due to the lack of TDP 43 function than to the toxicity of the aggregates.
Assuntos
Proteínas de Ligação a DNA/genética , Drosophila/metabolismo , Atividade Motora/genética , Neurônios Motores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Alelos , Animais , Animais Geneticamente Modificados , Comportamento Animal , Proteínas de Ligação a DNA/metabolismo , Humanos , FenótipoRESUMO
TDP-43 (also known as TARDBP) regulates different processes of gene expression, including transcription and splicing, through RNA and DNA binding. Moreover, recent reports have shown that the protein interacts with the 3'UTRs of specific mRNAs. The aberrant cellular distribution and aggregation of TDP-43 were recently reported in neurodegenerative diseases, namely frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A detailed description of the determinants for cellular localization has yet to emerge, including information on how the known functions of TDP-43 and cellular targeting affect each other. We provide the first experimental evidence that TDP-43 continuously shuttles between nucleus and cytoplasm in a transcription-dependent manner. Furthermore, we investigate the role of the functional TDP-43 domains in determining cellular targeting through a combination of immunofluorescence and biochemical fractionation methods. Our analyses indicate that the C-terminus is essential for solubility and cellular localization, because its deletion results in the formation of large nuclear and cytoplasmic aggregates. Disruption of the RNA-recognition domain required for RNA and DNA binding, however, alters nuclear distribution by decreasing TDP-43 presence in the nucleoplasm. Our findings suggest that TDP-43 solubility and localization are particularly sensitive to disruptions that extend beyond the newly found nuclear localization signal and depend on a combination of factors that are closely connected to the functional properties of this protein.