RESUMEN
In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5' end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5' end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.
Asunto(s)
Microscopía por Crioelectrón , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/química , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Conformación Proteica , Unión ProteicaRESUMEN
Translation of messenger ribonucleic acids (mRNAs) encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC-SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC-SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eukaryotic translation initiation factor 4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC-RNC-SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC-RNC-SRP. The NMD occurs only after the CBC-RNC-SRP is targeted to the ER and after eukaryotic translation initiation factor 4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC-RNC-SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.
Asunto(s)
Retículo Endoplásmico , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero , Partícula de Reconocimiento de Señal , Retículo Endoplásmico/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Humanos , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/genética , Señales de Clasificación de Proteína/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Células HeLa , Ribosomas/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Biosíntesis de ProteínasRESUMEN
We intend to evaluate the importance of N7 -methylguanosine (m7G) for the prognosis of breast cancer (BC). We gained 29 m7G-related genes from the published literature and among them, 16 m7G-related genes were found to have differential expression. Five differentially expressed genes (CYFIP1, EIF4E, EIF4E3, NCBP1 and WDR4) were linked to overall survival. This suggests that m7G-related genes might be prognostic or therapeutic targets for BC patients. We put the five genes to LASSO regression analysis to create a four-gene signature, including EIF4E, EIF4E3, WDR4 and NCBP1, that divides samples into two risky groups. Survival was drastically worsened in a high-risk group (p < 0.001). The signature's predictive capacity was demonstrated using ROC (10-year AUC 0.689; 10-year AUC 0.615; 3-year AUC 0.602). We found that immune status was significantly different between the two risk groups. In particular, NCBP1 also has a poor prognosis, with higher diagnostic value in ROC. NCBP1 also has different immune states according to its high or low expression. Meanwhile, knockdown of NCBP1 suppresses BC malignancy in vitro. Therefore, m7G RNA regulators are crucial participants in BC and four-gene mRNA levels are important predictors of prognosis. NCBP1 plays a critical target of m7G mechanism in BC.
Asunto(s)
Neoplasias de la Mama , Guanosina , Femenino , Humanos , Biomarcadores , Neoplasias de la Mama/genética , Factor 4E Eucariótico de Iniciación , Proteínas de Unión al GTP , Guanosina/análogos & derivados , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , PronósticoRESUMEN
In eukaryotic cells, RNAs transcribed by RNA polymerase-II receive the modification at the 5' end. This structure is called the cap structure. The cap structure has a fundamental role for translation initiation by recruiting eukaryotic translation initiation factor 4F (eIF4F). The other important mediator of the cap structure is a nuclear cap-binding protein complex (CBC). CBC consists of two proteins, which are renamed as NCBP1 and NCBP2 (previously called as CBP80/NCBP and CBP20/NIP1, respectively). This review article discusses the multiple roles CBC mediates and co-ordinates in several gene expression steps in eukaryotes.
Asunto(s)
Caperuzas de ARN , ARN Polimerasa II , Caperuzas de ARN/química , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Polimerasa II/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Complejo Proteico Nuclear de Unión a la Caperuza/química , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Células Eucariotas/metabolismoRESUMEN
Aberrant N7 -methylguanosine (m7G) levels closely correlate with tumor genesis and progression. NCBP2 and EIF4E3 are two important m7G-related cap-binding genes. This study aimed to identify the relationship between the EIF4E3/NCBP2 function and immunological characteristics of head and neck squamous cell carcinoma (HNSCC). Hierarchical clustering was employed in classifying HNSCC patients into two groups based on the expressions of NCBP2 and EIF4E3. The differentially expressed genes were identified between the two groups, and GO functional enrichment was subsequently performed. Weighted gene co-expression network analysis was conducted to identify the hub genes related to EIF4E3/NCBP2 expression and immunity. The differential infiltration of immune cells and the response to immunotherapy were compared between the two groups. Single-cell sequence and trajectory analyses were performed to predict cell differentiation and display the expression of EIF4E3/NCBP2 in each state. In addition, quantitative real-time PCR, spatial transcriptome analysis, transwell assay, and western blotting were conducted to verify the biological function of EIF4E3/NCBP2. Here, group A showed a higher EIF4E3 expression and a lower NCBP2 expression, which had higher immune scores, proportion of most immune cells, immune activities, expression of immunomodulatory targets, and a better response to cancer immunotherapy. Besides, 56 hub molecules with notable immune regulation significance were identified. A risk model containing 17 hub genes and a prognostic nomogram was successfully established. Moreover, HNSCC tissues had a lower EIF4E3 expression and a higher NCBP2 expression than normal tissues. NCBP2 and EIF4E3 played a vital role in the differentiation of monocytes. Furthermore, the expression of CCL4/CCL5 can be regulated via EIF4E3 overexpression and NCBP2 knockdown. Collectively, NCBP2 and EIF4E3 can affect downstream gene expression, as well as immune contexture and response to immunotherapy, which could induce "cold-to-hot" tumor transformation in HNSCC patients.
Asunto(s)
Quimiocina CCL4 , Quimiocina CCL5 , Factor 4E Eucariótico de Iniciación , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/fisiopatología , Neoplasias de Cabeza y Cuello/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/fisiopatología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Quimiocina CCL4/genética , Quimiocina CCL4/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Línea Celular Tumoral , Masculino , Femenino , Persona de Mediana Edad , Anciano , Inmunoterapia , Modelos Estadísticos , Mutación/genéticaRESUMEN
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.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Partícula de Reconocimiento de Señal/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Mitocondriales/genética , Modelos Genéticos , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/genética , Transducción de Señal/genéticaRESUMEN
Hypoxia as a crucial pathogenesis factor usually results in huge harmful effects on cardiac injury and dysfunction. Our previous study has uncovered the global transcriptome and translatome profiles of cardiomyocytes in vitro and in vivo to response to hypoxia by RNA sequencing and ribosome profiling sequencing. We observe a series of differential expressed genes between transcription and translation, which may be attributed to the hypoxia-specific binding affinity of nuclear cap-binding subunit 3 (NCBP3) at 5' untranslation region of target genes. Although we observe that NCBP3 can facilitate translational process in myocardium under hypoxia stress, the underlying molecular mechanism of NCBP3 for gene translation modulation remains unclear. In this study, we performed NCBP3 immunoprecipitation for mass spectrum and found that METTL3 and eIF4A2 particularly interacted with NCBP3 in hypoxic rat H9C2 cardiomyocytes. Furthermore, we observed that METTL3-mediated N6-methyladenosine (m6A) methylation was elevated in hypoxia, but compromised by NCBP3 or METTL3 knockdown. Finally, we also demonstrated that NCBP3/METTL3/eIF4A2 regulatory axis plays a specific role in cardiomyocytes undergoing hypoxic stress. Taken together, we unmasked NCBP3, a novel hypoxia-specific response protein functions as a scaffold to coordinate METTL3 and eIF4A2 for enhancing gene translation by m6A RNA methylation in cardiomyocytes upon hypoxic stress.
Asunto(s)
Hipoxia de la Célula , Metiltransferasas/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , ARN Mensajero/metabolismo , Animales , Línea Celular Tumoral , Metilación , Miocitos Cardíacos , RatasRESUMEN
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.
Asunto(s)
Encéfalo/embriología , Cromosomas Humanos Par 3/genética , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Discapacidad Intelectual/genética , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Proteínas de Xenopus/genética , Animales , Apoptosis/genética , Encéfalo/patología , Ciclo Celular/genética , Deleción Cromosómica , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Humanos , Discapacidad Intelectual/patología , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Lung cancer is the most frequent cancer type and is the leading cause of tumour-associated deaths worldwide. Nuclear cap-binding protein 1 (NCBP1) is necessary for capped RNA processing and intracellular localization. It has been reported that silencing of NCBP1 resulted in cell growth reduction in HeLa cells. Nevertheless, its clinical significance and underlying molecular mechanisms in non-small-cell lung cancer remain unclear. In this study, we found that NCBP1 was significantly overexpressed in lung cancer tissues and several lung cancer cell lines. Through knockdown and overexpression experiments, we showed that NCBP1 promoted lung cancer cell growth, wound healing ability, migration and epithelial-mesenchymal transition. Mechanistically, we found that cullin 4B (CUL4B) was a downstream target gene of NCBP1 in NSCLC. NCBP1 up-regulated CUL4B expression via interaction with nuclear cap-binding protein 3 (NCBP3). CUL4B silencing significantly reversed NCBP1-induced tumorigenesis in vitro. Based on these findings, we propose a model involving the NCBP1-NCBP3-CUL4B oncoprotein axis, providing novel insight into how CUL4B is activated and contributes to LUAD progression.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Carcinogénesis/patología , Proteínas Cullin/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Regulación hacia Arriba/genética , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Proteínas Cullin/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cicatrización de HeridasRESUMEN
The Paip2 protein is a factor regulating mRNA translation and stability in the cytoplasm. It has also been found in the nuclei of several cell types in Drosophila. Here, we aim to elucidate the functions of Paip2 in the cell nucleus. We find that nuclear Paip2 is a component of an ~300-kDa protein complex. Paip2 interacts with mRNA capping factor and factors of RNA polymerase II (Pol II) transcription initiation and early elongation. Paip2 functionally cooperates with the Cbp80 subunit of the cap-binding complex, with both proteins ensuring proper Pol II C-terminal domain (CTD) Ser5 phosphorylation at the promoter. Thus, Paip2 is a novel player at the stage of mRNA capping and early Pol II elongation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Animales , Línea Celular , ADN/metabolismo , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5' end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Saccharomyces cerevisiae Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing of SUS1 mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.
Asunto(s)
Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas de Unión a Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/fisiología , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Proteínas Nucleares/metabolismo , Empalme del ARN , Transporte de ARN/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
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.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Manihot/inmunología , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Enfermedades de las Plantas/inmunología , Potyviridae/inmunología , Sistemas CRISPR-Cas , Factor 4E Eucariótico de Iniciación/metabolismo , Edición Génica , Interacciones Huésped-Patógeno , Manihot/genética , Manihot/virología , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Enfermedades de las Plantas/prevención & control , Enfermedades de las Plantas/virología , Inmunidad de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Factor 4A Eucariótico de Iniciación/genética , VIH-1/genética , Complejo Proteico Nuclear de Unión a la Caperuza/genética , ARN Mensajero/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Línea Celular , Factor 4A Eucariótico de Iniciación/metabolismo , VIH-1/metabolismo , Células HeLa , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Unión Proteica , Empalme del ARN , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Replicación Viral/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Although peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) is a well-established transcriptional coactivator for the metabolic adaptation of mammalian cells to diverse physiological stresses, the molecular mechanism by which it functions is incompletely understood. Here we used in vitro binding assays, X-ray crystallography, and immunoprecipitations of mouse myoblast cell lysates to define a previously unknown cap-binding protein 80 (CBP80)-binding motif (CBM) in the C terminus of PGC-1α. We show that the CBM, which consists of a nine-amino-acid α helix, is critical for the association of PGC-1α with CBP80 at the 5' cap of target transcripts. Results from RNA sequencing demonstrate that the PGC-1α CBM promotes RNA synthesis from promyogenic genes. Our findings reveal a new conduit between DNA-associated and RNA-associated proteins that functions in a cap-binding protein surveillance mechanism, without which efficient differentiation of myoblasts to myotubes fails to occur.
Asunto(s)
Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/química , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Activación Transcripcional , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Diferenciación Celular , Humanos , Células MCF-7 , Ratones , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Caperuzas de ARN/metabolismo , Proteínas de Unión al ARN , Transcripción GenéticaRESUMEN
ARS2 is a highly conserved metazoan protein involved in numerous aspects of nuclear RNA metabolism. As a direct partner of the nuclear cap-binding complex (CBC), it mediates interactions with diverse RNA processing and transport machineries in a transcript-dependent manner. Here, we present the human ARS2 crystal structure, which exhibits similarities and metazoan-specific differences to the plant homologue SERRATE, most notably an additional RRM domain. We present biochemical, biophysical and cellular interactome data comparing wild type and mutant ARS2 that identify regions critical for interactions with FLASH (involved in histone mRNA biogenesis), NCBP3 (a putative cap-binding protein involved in mRNA export) and single-stranded RNA. We show that FLASH and NCBP3 have overlapping binding sites on ARS2 and that CBC-ARS2-NCBP3 form a ternary complex that is mutually exclusive with CBC-ARS-PHAX (involved in snRNA export). Our results support that mutually exclusive higher-order CBC-ARS2 complexes are critical in determining Pol II transcript fate.
Asunto(s)
Proteínas Nucleares/química , Transporte de ARN/fisiología , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Transcripción Genética/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Sitios de Unión/genética , Proteínas de Unión al Calcio/metabolismo , Cristalografía por Rayos X , Humanos , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas Nucleares/fisiología , Dominios Proteicos , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Nuclear RNA metabolism is influenced by protein complexes connecting to both RNA-productive and -destructive pathways. The ZC3H18 protein binds the cap-binding complex (CBC), universally present on capped RNAs, while also associating with the nuclear exosome targeting (NEXT) complex, linking to RNA decay. To dissect ZC3H18 function, we conducted interaction screening and mutagenesis of the protein, which revealed a phosphorylation-dependent isoform. Surprisingly, the modified region of ZC3H18 associates with core histone proteins. Further examination of ZC3H18 function, by genome-wide analyses, demonstrated its impact on transcription of a subset of protein-coding genes. This activity requires the CBC-interacting domain of the protein, with some genes being also dependent on the NEXT- and/or histone-interacting domains. Our data shed light on the domain requirements of a protein positioned centrally in nuclear RNA metabolism, and they suggest that post-translational modification may modulate its function.
Asunto(s)
Núcleo Celular/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Estabilidad del ARN/fisiología , Proteínas de Unión al ARN/metabolismo , ARN/biosíntesis , Núcleo Celular/química , Núcleo Celular/genética , Estudio de Asociación del Genoma Completo , Células HEK293 , Células HeLa , Humanos , Mutagénesis , Complejo Proteico Nuclear de Unión a la Caperuza/química , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Dominios Proteicos , ARN/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Pol II transcribes diverse classes of RNAs that need to be directed into the appropriate nuclear maturation pathway. All nascent Pol II transcripts are 5'-capped and the cap is immediately sequestered by the nuclear cap-binding complex (CBC). Mutually exclusive interactions of CBC with different partner proteins have been implicated in transcript fate determination. Here, we characterise the direct interactions between CBC and NELF-E, a subunit of the negative elongation factor complex, ARS2 and PHAX. Our biochemical and crystal structure results show that the homologous C-terminal peptides of NELF-E and ARS2 bind identically to CBC and in each case the affinity is enhanced when CBC is bound to a cap analogue. Furthermore, whereas PHAX forms a complex with CBC and ARS2, NELF-E binding to CBC is incompatible with PHAX binding. We thus define two mutually exclusive complexes CBC-NELF-E and CBC-ARS2-PHAX, which likely act in respectively earlier and later phases of transcription.
Asunto(s)
Complejo Proteico Nuclear de Unión a la Caperuza/química , Proteínas Nucleares/química , Factores de Transcripción/química , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Análogos de Caperuza de ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous in vitro studies, ALYREF binds to a region near the 5' end of the mRNA in a CBP80-dependent manner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3' end of the mRNA. Furthermore, the 3' processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5' and the 3' regions of the mRNA in vivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3' processing.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Factor de Estimulación del Desdoblamiento/genética , Factor de Estimulación del Desdoblamiento/metabolismo , Células HeLa , Humanos , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas Nucleares/genética , Proteína I de Unión a Poli(A)/metabolismo , Transporte de ARN , ARN Mensajero/química , Proteínas de Unión al ARN/genética , Factores de Transcripción/genéticaRESUMEN
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.