RESUMO
Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.
Assuntos
RNA Helicases DEAD-box/genética , Fatores de Iniciação em Eucariotos/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Biossíntese de Proteínas , Citoplasma/genética , Células HeLa , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Mapas de Interação de Proteínas/genética , Proteínas de Ligação ao Cap de RNA/genética , RNA Mensageiro/genéticaRESUMO
The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC-SRP interaction safeguards against abnormal expression of polypeptides from a ribosome-nascent chain complex (RNC)-SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC-SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Partícula de Reconhecimento de Sinal/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Mitocondriais/genética , Modelos Genéticos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/genética , Transdução de Sinais/genéticaRESUMO
The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development.
Assuntos
Encéfalo/embriologia , Cromossomos Humanos Par 3/genética , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Deficiência Intelectual/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Xenopus/genética , Animais , Apoptose/genética , Encéfalo/patologia , Ciclo Celular/genética , Deleção Cromossômica , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Deficiência Intelectual/patologia , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
A 5',7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Interest in the CBC has recently renewed due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosome. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative CBC together with NCBP1, and to interact with the canonical CBC along with the protein SRRT. The theme of post-transcriptional RNA fate, and how it relates to co-transcriptional ribonucleoprotein assembly, is abundant with complicated, ambiguous, and likely incomplete models. In an effort to clarify the compositions of NCBP1-, 2- and 3-related macromolecular assemblies, we have applied an affinity capture-based interactome screen where the experimental design and data processing have been modified to quantitatively identify interactome differences between targets under a range of experimental conditions. This study generated a comprehensive view of NCBP-protein interactions in the ribonucleoprotein context and demonstrates the potential of our approach to benefit the interpretation of complex biological pathways.
Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas Nucleares/genética , Proteoma/genética , Proteínas de Ligação ao Cap de RNA/genética , Citoplasma/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Humanos , Proteômica/métodos , Capuzes de RNA/genética , RNA Polimerase II/genéticaRESUMO
The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5'cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.
Assuntos
RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Transcrição Gênica , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Éxons , Regulação da Expressão Gênica/genética , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Ligação ao Cap de RNA/genética , RNA Polimerase II/genética , Estabilidade de RNA/genética , Transporte de RNA/genética , Fatores de Transcrição/genéticaRESUMO
ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5'cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.
Assuntos
Proteínas Nucleares/genética , RNA Helicases/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Células HEK293 , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas Nucleares/química , Domínios Proteicos/genética , RNA Helicases/química , RNA Mensageiro/química , RNA Nuclear/química , RNA Nuclear/genéticaRESUMO
Newly synthesized mRNAs are exported from the nucleus to cytoplasm with a 5'-cap structure bound by the nuclear cap-binding complex (CBC). During or after export, the CBC should be properly replaced by cytoplasmic cap-binding protein eIF4E for efficient protein synthesis. Nonetheless, little is known about how the replacement takes place. Here, we show that double-stranded RNA-binding protein staufen1 (STAU1) promotes efficient replacement by facilitating an association between the CBC-importin α complex and importin ß. Our transcriptome-wide analyses and artificial tethering experiments also reveal that the replacement occurs more efficiently when an mRNA associates with STAU1. This event is inhibited by a key nonsense-mediated mRNA decay factor, UPF1, which directly interacts with STAU1. Furthermore, we find that cellular apoptosis that is induced by ionizing radiation is accompanied by inhibition of the replacement via increased association between STAU1 and hyperphosphorylated UPF1. Altogether, our data highlight the functional importance of STAU1 and UPF1 in the course of the replacement of the CBC by eIF4E, adding a previously unappreciated layer of post-transcriptional gene regulation.
Assuntos
Proteínas do Citoesqueleto/genética , Fator de Iniciação 4E em Eucariotos/genética , Biossíntese de Proteínas/genética , RNA Helicases/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Regiões 5' não Traduzidas , Núcleo Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Ligação ao Cap de RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genéticaRESUMO
Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.
Assuntos
Fator de Iniciação 4A em Eucariotos/genética , HIV-1/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , RNA Mensageiro/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Linhagem Celular , Fator de Iniciação 4A em Eucariotos/metabolismo , HIV-1/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Ligação Proteica , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.
Assuntos
Fator de Iniciação 4E em Eucariotos/genética , Manihot/imunologia , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Doenças das Plantas/imunologia , Potyviridae/imunologia , Sistemas CRISPR-Cas , Fator de Iniciação 4E em Eucariotos/metabolismo , Edição de Genes , Interações Hospedeiro-Patógeno , Manihot/genética , Manihot/virologia , Complexo Proteico Nuclear de Ligação ao Cap/genética , Doenças das Plantas/prevenção & controle , Doenças das Plantas/virologia , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
RNA turnover is necessary for controlling proper mRNA levels posttranscriptionally. In general, RNA degradation is via exoribonucleases that degrade RNA either from the 5' end to the 3' end, such as XRN4, or in the opposite direction by the multisubunit exosome complex. Here, we use genome-wide mapping of uncapped and cleaved transcripts to reveal the global landscape of cotranslational mRNA decay in the Arabidopsis thaliana transcriptome. We found that this process leaves a clear three nucleotide periodicity in open reading frames. This pattern of cotranslational degradation is especially evident near the ends of open reading frames, where we observe accumulation of cleavage events focused 16 to 17 nucleotides upstream of the stop codon because of ribosomal pausing during translation termination. Following treatment of Arabidopsis plants with the translation inhibitor cycloheximide, cleavage events accumulate 13 to 14 nucleotides upstream of the start codon where initiating ribosomes have been stalled with these sequences in their P site. Further analysis in xrn4 mutant plants indicates that cotranslational RNA decay is XRN4 dependent. Additionally, studies in plants lacking CAP BINDING PROTEIN80/ABA HYPERSENSITIVE1, the largest subunit of the nuclear mRNA cap binding complex, reveal a role for this protein in cotranslational decay. In total, our results demonstrate the global prevalence and features of cotranslational RNA decay in a plant transcriptome.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , Arabidopsis/efeitos dos fármacos , Cicloeximida/farmacologia , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estabilidade de RNA/fisiologia , RNA de Plantas/genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5' splice site of a pre-mRNA and the 5' end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5'-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences. This led to the unexpected findings that the AU dinucleotide dictates the optimal binding of cap-binding complex (CBC) to the 5' end of the nascent U1 snRNA, which ultimately influences the utilization of U1 snRNP in splicing. Our data also provide a structural interpretation as to why the AU dinucleotide is conserved during evolution.
Assuntos
Proteínas de Ligação ao Cap de RNA/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Pareamento de Bases , Simulação de Acoplamento Molecular , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras/genética , Leveduras/crescimento & desenvolvimentoRESUMO
Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that in mammals generally occurs upon recognition of a premature termination codon (PTC) during a pioneer round of translation. This round involves newly synthesized mRNA that is bound at its 5' end by the cap-binding protein (CBP) heterodimer CBP80-CBP20. Here we show that precluding the binding of the NMD factor UPF1 to CBP80 inhibits NMD at two steps: the association of SMG1 and UPF1 with the two eukaryotic release factors (eRFs) during SURF complex formation at a PTC, and the subsequent association of SMG1 and UPF1 with an exon-junction complex. We also demonstrate that UPF1 binds PTC-containing mRNA more efficiently than the corresponding PTC-free mRNA in a way that is promoted by the UPF1-CBP80 interaction. A unifying model proposes a choreographed series of protein-protein interactions occurring on an NMD target.
Assuntos
Códon sem Sentido , Modelos Biológicos , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Células HeLa , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , RNA Helicases , RNA Mensageiro/genética , Transativadores/genéticaRESUMO
The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.
Assuntos
Sequência Conservada , Proteínas Nucleares/genética , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos , Transporte Biológico , Clonagem Molecular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
mRNA transport from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. A pre-mRNA molecule undergoes modification, such as 5'-capping, splicing, and 3'-end processing, in the nucleus. The molecule being modified interacts with a large number of proteins and, thus, mRNP particles are formed. The binding of factors involved in nuclear export also occurs during transcription and mRNA processing. We have shown that the functioning of TREX-2, an mRNA export complex, is restricted to the nucleus. We used the method of RNA coprecipitation that enables the selective extraction of RNA-protein complexes from samples to show that the transcription elongation complex TREX interacts with mRNA of the ß-tubulin 56D gene over the entire length of the molecule. The capping protein Cbp80 reacted both with the cap structure and with a specific part of the coding mRNA of the ß-tubulin 56D gene. The TREX-2 complex that mediates mRNA export from the nucleus to the cytoplasm is bound to the same part of the coding sequence. Thus, we identified a common binding site for all of the complexes under investigation on the mRNA of ß-tubulin 56D. Co-immunoprecipitation reactions performed with S2 cell extracts revealed interactions between the components of complexes involved in transcription elongation, maturation, and export of mRNA. The model of molecular folding for the mRNP particle involving the mRNA of ß-tubulin 56D has been proposed.
Assuntos
Modelos Biológicos , Complexos Multiproteicos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Elongação da Transcrição Genética/fisiologia , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Complexos Multiproteicos/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Tubulina (Proteína)/genéticaRESUMO
Multiple endocrine neoplasia type I (MEN1) is an inherited syndrome that includes susceptibility to pancreatic islet hyperplasia. This syndrome results from mutations in the MEN1 gene, which encodes menin protein. Menin interacts with several transcription factors, including JunD, and inhibits their activities. However, the precise mechanism by which menin suppresses gene expression is not well understood. Here, we show that menin interacts with arsenite-resistant protein 2 (ARS2), a component of the nuclear RNA CAP-binding complex that is crucial for biogenesis of certain miRNAs including let-7a. The levels of primary-let-7a (pri-let-7a) are not affected by menin; however, the levels of mature let-7a are substantially decreased upon Men1 excision. Let-7a targets, including Insr and Irs2, pro-proliferative genes that are crucial for insulin-mediated signaling, are up-regulated in Men1-excised cells. Inhibition of let-7a using anti-miRNA in wild type cells is sufficient to enhance the expression of insulin receptor substrate 2 (IRS2) to levels observed in Men1-excised cells. Depletion of menin does not affect the expression of Drosha and CBP80, but substantially impairs the processing of pri-miRNA to pre-miRNA. Ars2 knockdown decreased let-7a processing in menin-expressing cells but had little impact on let-7a levels in menin-excised cells. As IRS2 is known to mediate insulin signaling and insulin/mitogen-induced cell proliferation, these findings collectively unravel a novel mechanism whereby menin suppresses cell proliferation, at least partly by promoting the processing of certain miRNAs, including let-7a, leading to suppression of Irs2 expression and insulin signaling.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Processamento Pós-Transcricional do RNA , RNA Neoplásico/biossíntese , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , MicroRNAs/genética , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/patologia , Proteínas de Neoplasias/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Neoplásico/genética , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Ribonuclease III/biossíntese , Ribonuclease III/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3'-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC's role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly.
Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Spliceossomos/metabolismo , Sítios de Ligação , Genes fos , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Complexo Proteico Nuclear de Ligação ao Cap/química , Complexo Proteico Nuclear de Ligação ao Cap/genética , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U5/químicaRESUMO
The 7mG (7-methylguanosine cap) formed on mRNA is fundamental to eukaryotic gene expression. Protein complexes recruited to 7mG mediate key processing events throughout the lifetime of the transcript. One of the most important mediators of 7mG functions is CBC (cap-binding complex). CBC has a key role in several gene expression mechanisms, including transcription, splicing, transcript export and translation. Gene expression can be regulated by signalling pathways which influence CBC function. The aim of the present review is to discuss the mechanisms by which CBC mediates and co-ordinates multiple gene expression events.
Assuntos
Guanosina/análogos & derivados , Proteínas de Ligação ao Cap de RNA/metabolismo , Capuzes de RNA/metabolismo , Animais , Regulação da Expressão Gênica , Guanosina/química , Guanosina/genética , Guanosina/metabolismo , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/química , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas de Ligação ao Cap de RNA/química , Proteínas de Ligação ao Cap de RNA/genética , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genéticaRESUMO
Yeast pre-mRNA splicing initiates via formation of a complex comprising U1 snRNP bound at the 5' splice site (5'SS) and the Msl5â¢Mud2 heterodimer engaged at the branchpoint (BP). Here, we present a mutational analysis of the U1 snRNA, which shows that although enlarging the 5' leader between the TMG cap and the (3)ACUUAC(8) motif that anneals to the 5'SS is tolerated, there are tight constraints on the downstream spacer between (3)ACUUAC(8) and helix 1 of the U1 fold. We exploit U1 alleles with 5' extensions, variations in the (3)ACUUAC(8) motif, downstream mutations and a longer helix 1 to discover new intra-snRNP synergies with U1 subunits Nam8 and Mud1 and the trimethylguanosine (TMG) cap. We describe novel mutations in U1 snRNA that bypass the essentiality of the DEAD-box protein Prp28. Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential. We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2. Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNPâ¢5'SS complex with the Mud2â¢Msl5â¢BP complex.
Assuntos
RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alelos , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Elementos de DNA Transponíveis , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Mapeamento de Interação de Proteínas , Fatores de Processamento de RNA , RNA Fúngico/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Spliceossomos/genética , Spliceossomos/metabolismo , Fator de Processamento U2AF , Relação Estrutura-AtividadeRESUMO
The cap-binding proteins CBP20 and CBP80 have well-established roles in RNA metabolism and plant growth and development. Although these proteins are thought to be involved in the plant's response to environmental stress, their functions in this process are unclear. Here we demonstrated that Arabidopsis cbp20 and cbp80 null mutants had abnormal leaves and flowers and exhibited increased sensitivity to salt stress. The aberrant phenotypes were more pronounced in the cbp20/80 double mutant. Quantification by iTRAQ (isobaric tags for relative and absolute quantification) identified 77 differentially expressed proteins in the cbp20 and cbp80 lines compared with the wild-type Col-0 under salt stress conditions. Most of these differentially expressed proteins were synergistically expressed in cbp20 and cbp80, suggesting that CBP20 and CBP80 have synergistic roles during the salt stress response. Biochemical analysis demonstrated that CBP20 and CBP80 physically interacted with each other. Further analysis revealed that CBP20/80 regulated the splicing of genes involved in proline and sugar metabolism and that the epigenetic and post-translational modifications of these genes were involved in salt stress tolerance. Our data suggest a link between CBP20/80-dependent protein ubiquitination/sumoylation and the salt stress response.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/metabolismo , Tolerância ao Sal/fisiologia , Processamento Alternativo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Flores/genética , Flores/metabolismo , Flores/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Immunoblotting , Mutação , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância ao Sal/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MicroRNA (miRNA) biogenesis proceeds from a primary transcript (pri-miRNA) through the pre-miRNA into the mature miRNA. Here, we identify a role of the Caenorhabditis elegans nuclear export receptor XPO-1 and the cap-binding proteins CBP-20/NCBP-2 and CBP-80/NCBP-1 in this process. The RNA-mediated interference of any of these genes causes retarded heterochronic phenotypes similar to those observed for animals with mutations in the let-7 miRNA or core miRNA machinery genes. Moreover, pre- and mature miRNAs become depleted, whereas primary miRNA transcripts accumulate. An involvement of XPO-1 in miRNA biogenesis is conserved in Drosophila, in which knockdown of Embargoed/XPO-1 or its chemical inhibition through leptomycin B causes pri-miRNA accumulation. Our findings demonstrate that XPO-1/Emb promotes the pri-miRNA-to-pre-miRNA processing and we propose that this function involves intranuclear transport and/or nuclear export of primary miRNAs.