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1.
Cell ; 170(2): 312-323.e10, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28708999

RESUMO

Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Proteínas do Citoesqueleto/química , Humanos , Camundongos , Domínios Proteicos , Splicing de RNA , Alinhamento de Sequência , Fatores de Processamento de Serina-Arginina/metabolismo
2.
Mol Cell ; 84(8): 1496-1511.e7, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38537639

RESUMO

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.


Assuntos
Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Animais , Spliceossomos/genética , Spliceossomos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Íntrons/genética , Cromatina/genética , Cromatina/metabolismo , Splicing de RNA , Precursores de RNA/metabolismo , Mamíferos/metabolismo
3.
Cell ; 165(3): 606-19, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-27104978

RESUMO

Rbfox proteins control alternative splicing and posttranscriptional regulation in mammalian brain and are implicated in neurological disease. These proteins recognize the RNA sequence (U)GCAUG, but their structures and diverse roles imply a variety of protein-protein interactions. We find that nuclear Rbfox proteins are bound within a large assembly of splicing regulators (LASR), a multimeric complex containing the proteins hnRNP M, hnRNP H, hnRNP C, Matrin3, NF110/NFAR-2, NF45, and DDX5, all approximately equimolar to Rbfox. We show that splicing repression mediated by hnRNP M is stimulated by Rbfox. Virtually all the intron-bound Rbfox is associated with LASR, and hnRNP M motifs are enriched adjacent to Rbfox crosslinking sites in vivo. These findings demonstrate that Rbfox proteins bind RNA with a defined set of cofactors and affect a broader set of exons than previously recognized. The function of this multimeric LASR complex has implications for deciphering the regulatory codes controlling splicing networks.


Assuntos
Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Éxons , Células HEK293 , Humanos , Íntrons , Camundongos , Complexos Multiproteicos/metabolismo , Precursores de RNA/metabolismo
5.
Nature ; 587(7832): 145-151, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32908311

RESUMO

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear1. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus2 and binding diverse proteins3-5 to achieve X-chromosome inactivation (XCI)6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off8. Here we show that the Xist RNA-binding proteins PTBP19, MATR310, TDP-4311 and CELF112 assemble on the multivalent E-repeat element of Xist7 and, via self-aggregation and heterotypic protein-protein interactions, form a condensate1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase8, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.


Assuntos
Inativação Gênica , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas CELF1/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Inativação do Cromossomo X/genética
6.
bioRxiv ; 2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37790489

RESUMO

Understanding the mechanisms of pre-mRNA splicing and spliceosome assembly is limited by technical challenges to examining spliceosomes in vivo. Here we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. Sequencing these pre-mRNA fragments allowed the transcriptome-wide mapping of branch sites with high sensitivity. In addition to known U2 snRNP proteins, these complexes contained the proteins RBM5 and RBM10. RBM5 and RBM10 are alternative splicing regulators that control exons affecting apoptosis and cell proliferation in cancer, but were not previously shown to associate with the U2 snRNP or to play roles in branch site selection. We delineate a common segment of RBM5 and RBM10, separate from their known functional domains, that is required for their interaction with the U2 snRNP. We identify a large set of splicing events regulated by RBM5 and RBM10 and find that they predominantly act as splicing silencers. Disruption of their U2 interaction renders the proteins inactive for repression of many alternative exons. We further find that these proteins assemble on branch sites of nearly all exons across the transcriptome, including those whose splicing is not altered by them. We propose a model where RBM5 and RBM10 act as components of the U2 snRNP complex. From within this complex, they sense structural features of branchpoint recognition to either allow progression to functional spliceosome or rejection of the complex to inhibit splicing.

7.
RNA ; 16(2): 405-16, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20042473

RESUMO

The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxDeltaRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxDeltaRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform.


Assuntos
Processamento Alternativo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Feminino , Expressão Gênica , Homeostase , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/química , Homologia de Sequência de Aminoácidos
8.
Neuron ; 98(1): 127-141.e7, 2018 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-29621484

RESUMO

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.


Assuntos
Inibição Neural/fisiologia , Neurônios/metabolismo , Fatores de Processamento de RNA/fisiologia , Transmissão Sináptica/fisiologia , Proteína 1 Associada à Membrana da Vesícula/biossíntese , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/química , Fatores de Processamento de RNA/análise , Fatores de Processamento de RNA/deficiência , Proteínas SNARE/análise , Proteínas SNARE/biossíntese , Proteína 1 Associada à Membrana da Vesícula/análise
9.
Mol Cell Biol ; 24(4): 1700-8, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14749385

RESUMO

U12-dependent introns are spliced by the so-called minor spliceosome, requiring the U11, U12, and U4atac/U6atac snRNPs in addition to the U5 snRNP. We have recently identified U6-p110 (SART3) as a novel human recycling factor that is related to the yeast splicing factor Prp24. U6-p110 transiently associates with the U6 and U4/U6 snRNPs during the spliceosome cycle, regenerating functional U4/U6 snRNPs from singular U4 and U6 snRNPs. Here we investigated the involvement of U6-p110 in recycling of the U4atac/U6atac snRNP. In contrast to the major U6 and U4/U6 snRNPs, p110 is primarily associated with the U6atac snRNP but is almost undetectable in the U4atac/U6atac snRNP. Since p110 does not occur in U5 snRNA-containing complexes, it appears to be transiently associated with U6atac during the cycle of the minor spliceosome. The p110 binding site was mapped to U6 nucleotides 38 to 57 and U6atac nucleotides 10 to 30, which are highly conserved between these two functionally related snRNAs. With a U12-dependent in vitro splicing system, we demonstrate that p110 is required for recycling of the U4atac/U6atac snRNP.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Sequência de Bases , Sítios de Ligação , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/química , Spliceossomos/genética
10.
Methods Mol Biol ; 1648: 155-167, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28766296

RESUMO

Eukaryotic organelles or subcellular compartments can be selectively isolated based on their physical density and their stability in the presence of nonionic detergents. This chapter describes a protocol for the preparation of cytoplasm, nucleoplasm, and chromatin, in addition to isolation of RNA and proteins from these fractions. Proteins and protein complexes stably associated with chromatin and other high molecular weight nuclear components can be extracted under non-denaturing conditions by enzymatic digestion of RNA and DNA. The chapter also includes a detailed extraction protocol from highly purified nuclei.


Assuntos
Cromatina/química , Citoplasma/química , Complexos Multiproteicos , RNA , Animais , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , RNA/química , RNA/isolamento & purificação
11.
J Clin Invest ; 127(3): 987-1004, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28192372

RESUMO

A highly orchestrated gene expression program establishes the properties that define mature adipocytes, but the contribution of posttranscriptional factors to the adipocyte phenotype is poorly understood. Here we have shown that the RNA-binding protein PSPC1, a component of the paraspeckle complex, promotes adipogenesis in vitro and is important for mature adipocyte function in vivo. Cross-linking and immunoprecipitation followed by RNA sequencing revealed that PSPC1 binds to intronic and 3'-untranslated regions of a number of adipocyte RNAs, including the RNA encoding the transcriptional regulator EBF1. Purification of the paraspeckle complex from adipocytes further showed that PSPC1 associates with the RNA export factor DDX3X in a differentiation-dependent manner. Remarkably, PSPC1 relocates from the nucleus to the cytoplasm during differentiation, coinciding with enhanced export of adipogenic RNAs. Mice lacking PSPC1 in fat displayed reduced lipid storage and adipose tissue mass and were resistant to diet-induced obesity and insulin resistance due to a compensatory increase in energy expenditure. These findings highlight a role for PSPC1-dependent RNA maturation in the posttranscriptional control of adipose development and function.


Assuntos
Adipócitos/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células 3T3-L1 , Transporte Ativo do Núcleo Celular/genética , Adipócitos/patologia , Animais , Núcleo Celular/genética , Núcleo Celular/patologia , RNA Helicases DEAD-box , Metabolismo Energético/genética , Camundongos , Camundongos Knockout , Células NIH 3T3 , Proteínas Nucleares/genética , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismo
12.
Neuron ; 89(1): 113-28, 2016 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-26687839

RESUMO

Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3' UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease.


Assuntos
Transtorno Autístico/genética , Estriol/análogos & derivados , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Citoplasma/metabolismo , Estriol/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos Endogâmicos C57BL , Fatores de Processamento de RNA
13.
Elife ; 4: e09268, 2015 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-26705333

RESUMO

The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas de Homeodomínio/metabolismo , Neurônios/fisiologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Camundongos , Fator de Transcrição 1 de Leucemia de Células Pré-B
14.
Nat Genet ; 43(7): 706-11, 2011 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-21623373

RESUMO

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear. We show here that central nervous system-specific deletion of the gene encoding Rbfox1 results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording revealed a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole-transcriptome analyses identified multiple splicing changes in the Rbfox1(-/-) brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function.


Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/fisiologia , Animais , Apoptose , Biomarcadores/metabolismo , Western Blotting , Encéfalo/citologia , Proliferação de Células , Eletrofisiologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Processamento de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/induzido quimicamente , Transmissão Sináptica
15.
Nat Struct Mol Biol ; 15(2): 183-91, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18193060

RESUMO

The polypyrimidine tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.


Assuntos
Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Spliceossomos/química , Spliceossomos/metabolismo , Extratos Celulares , Linhagem Celular , Éxons , Humanos , Íntrons , Modelos Biológicos , Ligação Proteica , Proteínas/análise , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo
16.
Proc Natl Acad Sci U S A ; 104(16): 6608-13, 2007 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-17416673

RESUMO

The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Spliceossomos/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Genes Letais , Mutagênese , Especificidade de Órgãos/genética , Fenótipo , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Timo/anormalidades , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
17.
Biol Chem ; 387(10-11): 1455-60, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17081119

RESUMO

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.


Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Ligação Proteica , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética
18.
EMBO J ; 21(11): 2724-35, 2002 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-12032085

RESUMO

During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4-U6 and U6-U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as p110(nrb) or SART3. p110 contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri-snRNPs nor with spliceosomes. Recom binant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 snRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle.


Assuntos
Antígenos de Neoplasias , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/fisiologia , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/fisiologia , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Western Blotting , Núcleo Celular/metabolismo , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
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