RESUMEN
During spliceosome assembly, the 3' splice site is recognized by sequential U2AF2 complexes, first with Splicing Factor 1 (SF1) and second by the SF3B1 subunit of the U2 small nuclear ribonuclear protein particle. The U2AF2-SF1 interface is well characterized, comprising a U2AF homology motif (UHM) of U2AF2 bound to a U2AF ligand motif (ULM) of SF1. However, the structure of the U2AF2-SF3B1 interface and its importance for pre-mRNA splicing are unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8-Å resolution. We discovered a distinctive trajectory of the SF3B1 ULM across the U2AF2 UHM surface, which differs from prior UHM/ULM structures and is expected to modulate the orientations of the full-length proteins. We established that the binding affinity of the U2AF2 UHM for the cocrystallized SF3B1 ULM rivals that of a nearly full-length U2AF2 protein for an N-terminal SF3B1 region. An additional SF3B6 subunit had no detectable effect on the U2AF2-SF3B1 binding affinities. We further showed that key residues at the U2AF2 UHM-SF3B1 ULM interface contribute to coimmunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2-SF3B1 interface changed splicing of representative human transcripts. From analysis of genome-wide data, we found that many of the splice sites coregulated by U2AF2 and SF3B1 differ from those coregulated by U2AF2 and SF1. Taken together, these findings support distinct structural and functional roles for the U2AF2-SF1 and U2AF2-SF3B1 complexes during the pre-mRNA splicing process.
Asunto(s)
Precursores del ARN , Factores de Empalme de ARN/química , Empalme del ARN , Factor de Empalme U2AF/química , Humanos , Ligandos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , Factor de Empalme U2AF/metabolismoRESUMEN
The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD+ metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD+ biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss-Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD+ biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation.
Asunto(s)
NAD , Poli(ADP-Ribosa) Polimerasas , ADP-Ribosilación , Animales , Células Madre Embrionarias/metabolismo , Ratones , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , ProteómicaRESUMEN
In all organisms, splicing occurs through the formation of spliceosome complexes, and splicing auxiliary factors are essential during splicing. U2AF65 is a crucial splicing cofactor, and the two typical RNA-recognition motifs at its center recognize and bind the polypyrimidine sequence located between the intron branch site and the 3'-splice site. U2AF65A is a member of the U2AF65 gene family, with pivotal roles in diseases in mammals, specifically humans; however, few studies have investigated plant U2AF65A, and its specific functions are poorly understood. Therefore, in the present study, we systematically identified U2AF65A in plant species from algae to angiosperms. Based on 113 putative U2AF65A sequences from 33 plant species, phylogenetic analyses were performed, followed by basic bioinformatics, including the comparisons of gene structure, protein domains, promoter motifs, and gene expression levels. In addition, using rice as the model crop, we demonstrated that the OsU2AF65A protein is localized to the nucleus and cytoplasm, and it is involved in responses to various stresses, such as drought, high salinity, low temperature, and heavy metal exposure (e.g., cadmium). Using Arabidopsis thaliana and rice mutants, we demonstrated that U2AF65A is involved in the accumulation of plant biomass, growth of hypocotyl upon thermal stimulation, and reduction of tolerance of high temperature stress. These findings offer an overview of the U2AF65 gene family and its stress response functions, serving as the reference for further comprehensive functional studies of the essential specific splicing cofactor U2AF65A in the plant kingdom.
RESUMEN
Pre-mRNA splicing is a major process in the regulated expression of genes in eukaryotes, and alternative splicing is used to generate different proteins from the same coding gene. Splicing is a catalytic process that removes introns and ligates exons to create the RNA sequence that codifies the final protein. While this is achieved in an autocatalytic process in ancestral group II introns in prokaryotes, the spliceosome has evolved during eukaryogenesis to assist in this process and to finally provide the opportunity for intron-specific splicing. In the early stage of splicing, the RNA 5' and 3' splice sites must be brought within proximity to correctly assemble the active spliceosome and perform the excision and ligation reactions. The assembly of this first complex, termed E-complex, is currently the least understood process. We focused in this review on the formation of the E-complex and compared its composition and function in three different organisms. We highlight the common ancestral mechanisms in S. cerevisiae, S. pombe, and mammals and conclude with a unifying model for intron definition in constitutive and regulated co-transcriptional splicing.
Asunto(s)
Empalme Alternativo , Mamíferos/genética , Precursores del ARN/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Empalmosomas/genética , Animales , Secuencia de Bases , Evolución Molecular , Exones , Humanos , Intrones , Mamíferos/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Empalmosomas/química , Empalmosomas/metabolismo , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Splicing of mRNA precursors is essential in the regulation of gene expression. U2AF65 recognizes the poly-pyrimidine tract and helps in the recognition of the branch point. Inactivation of fission yeast U2AF65 (Prp2) blocks splicing of most, but not all, pre-mRNAs, for reasons that are not understood. Here, we have determined genome-wide the splicing efficiency of fission yeast cells as they progress into synchronous meiosis in the presence or absence of functional Prp2. Our data indicate that in addition to the splicing elements at the 3' end of any intron, the nucleotides immediately upstream the intron will determine whether Prp2 is required or dispensable for splicing. By changing those nucleotides in any given intron, we regulate its Prp2 dependency. Our results suggest a model in which Prp2 is required for the coordinated recognition of both intronic ends, placing Prp2 as a key regulatory element in the determination of the exon-intron boundaries.
Asunto(s)
Exones , Intrones , Empalme del ARN , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Factor de Empalme U2AF , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.
Asunto(s)
Precursores del ARN/efectos de los fármacos , Empalme del ARN/efectos de los fármacos , ARN Neoplásico/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Empalme U2AF/antagonistas & inhibidores , Femenino , Células HEK293 , Humanos , Células K562 , Simulación del Acoplamiento Molecular , Estructura Molecular , Precursores del ARN/genética , Empalme del ARN/genética , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Hsa_circ_001988 has been identified as a tumor suppressor gene in several carcinomas. However, its expression pattern and role in gastric cancer (GC) have still remained elusive. This study aimed to explore the functions of hsa_circ_001988 in GC. Quantitative reverse transcription polymerase chain reaction assay was performed to assess the expressions of hsa_circ_001988, miR-197-3p, FBXW7, CCDC6, and U2AF65 in GC tissues. The correlation analysis was undertaken to find out the relationship between hsa_circ_001988 expression and clinicopathological factors. A series of cellular experiments were carried out to describe the effects of hsa_circ_001988 on GC in vivo and in vitro. Besides, RNA immunoprecipitation (RIP) assay was performed to verify the relationship among EIF4A3, U2AF65, and hsa_circ_001988. We first found that the expression of hsa_circ_001988 was decreased in 341 GC patients that was related to World Health Organization histological types, Lauren types, and tumor invasion depth (p < .05). Silencing of hsa_circ_001988 facilitated proliferation, colony formation, migration, and invasion of GC cells, while overexpression of hsa_circ_001988 reversed the effect on GC progression in vitro. Additionally, the results of subcutaneous xenotransplanted tumor model demonstrated that overexpressing hsa_circ_001988 significantly suppressed the subcutaneous tumor growth in vivo. Mechanistically, hsa_circ_001988 attenuated the miR-197-3p expression possibly due to its molecular sponge effect, and then, positively promoted FBXW7 expression. Afterwards, FBXW7 regulated the expressions of yes-associated protein 1, cyclinD1, CCDC6, and EMT-related proteins. Notably, RIP assay showed the enrichment relationship among EIF4A3, U2AF65, and hsa_circ_001988. Additionally, EIF4A3 or U2AF65 promoted cyclization of hsa_circ_001988 in GC. Hsa_circ_001988 inhibits the proliferation and metastasis of GC via modulating EIF4A3/U2AF65-mediated hsa_circ_001988/miR-197-3p/FBXW7 axis.
Asunto(s)
MicroARNs/genética , ARN Circular/genética , Neoplasias Gástricas/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana EdadRESUMEN
High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.
Asunto(s)
Mutación Missense , Proteínas de Neoplasias , Neoplasias , Empalme del ARN , ARN Neoplásico , Factor de Empalme U2AF , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/química , Neoplasias/genética , Neoplasias/metabolismo , ARN Neoplásico/química , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Factor de Empalme U2AF/química , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Segment 8 mRNAs of influenza virus A/Brevig Misson/1918/1 (H1N1) are poorly spliced compared to segment 8 mRNAs of influenza virus A/Netherlands/178/95 (H3N2). Using oligonucleotide-mediated protein pull down with oligos spanning the entire length of segment 8 of either influenza virus H1N1 or influenza virus H3N2 we identified cellular RNA binding proteins that interacted with oligonucleotides derived from either H1N1 or H3N2 sequences. When the identified hot spots for RNA binding proteins in H1N1 segment 8 mRNAs were replaced by H3N2 sequences, splicing efficiency increased significantly. Replacing as few as three nucleotides of the H1N1 mRNA with sequences from H3N2 mRNA, enhanced splicing of the H1N1 mRNAs. Cellular proteins U2AF65 and HuR interacted preferentially with the 3'-splice site of H3N2 and overexpression of HuR reduced the levels of unspliced H1N1 mRNAs, suggesting that U2AF65 and HuR contribute to control of influenza virus mRNA splicing.
Asunto(s)
Empalme Alternativo , Proteína 1 Similar a ELAV/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , ARN Mensajero/genética , Factor de Empalme U2AF/genética , Células A549 , Proteína 1 Similar a ELAV/metabolismo , Variación Genética , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Factor de Empalme U2AF/metabolismo , Transfección , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Despite years of study, the gestational disorder preeclampsia (PE) remains poorly understood. One proposed mechanism of PE development is increased soluble VEGF receptor-1 (sFlt-1), ultimately causing angiogenic imbalance and endothelial dysfunction. The soluble protein is an alternative splice variant of FLT1, which also encodes for the full-length receptor Flt-1. The mechanism of the alternative splicing, and the reason for its inappropriate increase in preeclampsia, is not well understood. U2 auxiliary factor 65 (U2AF65) and jumonji C domain-containing protein 6 (JMJD6) have been implicated in the splicing of sFlt-1. Using siRNA knockdown and plasmid overexpression in immortalized placental trophoblasts (BeWo) and primary endothelial cells (HUVECs), we examined the role these proteins play in production of sFlt-1. Our results showed that U2AF65 has little, if any, effect on sFlt-1 splicing, and JMJD6 may enhance sFlt-1 splicing, but is not necessary for splicing to occur. Utilizing a hypoxic environment to mimic conditions of the preeclamptic placenta, as well as examining placentae in the reduced uterine perfusion pressure (RUPP) model of PE, which exhibits increased circulating sFlt-1, we found increased expression of JMJD6 in both hypoxic cells and placental tissue. Additionally, we observed a potential role for U2AF65 and JMJD6 to regulate the extracellular matrix enzyme heparanase, which may be involved in the release of sFlt-1 protein from the extracellular matrix. It will be important to study the role of these proteins in different tissues in the future, as changes in expression had differential effects on sFlt-1 splicing in the different cell types studied here.
Asunto(s)
Empalme Alternativo , Células Endoteliales de la Vena Umbilical Humana/enzimología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Preeclampsia/enzimología , Factor de Empalme U2AF/metabolismo , Trofoblastos/enzimología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Hipoxia de la Célula , Línea Celular , Modelos Animales de Enfermedad , Femenino , Glucuronidasa/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Preeclampsia/genética , Preeclampsia/fisiopatología , Embarazo , Ratas Sprague-Dawley , Factor de Empalme U2AF/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The function of U2AF65 in alternative splicing has been identified; however, the essential physiological role of U2AF65 remains poorly understood. In this study, we investigated the regulatory role of U2AF65 in milk synthesis and growth of bovine mammary epithelial cells (BMECs). Our results showed that U2AF65 localizes in the nucleus. Treatment with amino acids (Met and Leu) and hormones (prolactin and ß-estradiol) upregulated the expression of U2AF65 in these cells. U2AF65 overexpression increased the synthesis of ß-casein, triglycerides, and lactose; increased cell viability; and promoted proliferation of BMECs. Furthermore, our results showed that U2AF65 positively regulated mTOR phosphorylation and expression of mature mRNA of mTOR and SREBP-1c. Collectively, our findings demonstrate that U2AF65 regulates the mRNA expression of signalling molecules (mTOR and SREBP-1c) involved in milk synthesis and growth of BMECs, possibly via controlling the splicing and maturation of these mRNAs. U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The essential physiological role of U2AF65 remains poorly understood. In the present study, we confirmed that U2AF65 functions as a positive regulator of milk synthesis in and proliferation of bovine mammary epithelial cells via the mTOR-SREBP-1c signalling pathway. Therefore, our study uncovers the regulatory role of U2AF65 in milk synthesis and cell proliferation.
Asunto(s)
Proliferación Celular , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Transducción de Señal , Factor de Empalme U2AF/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Bovinos , Núcleo Celular/metabolismo , Células Epiteliales/citología , Femenino , Glándulas Mamarias Animales/citología , FosforilaciónRESUMEN
Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.
Asunto(s)
Papillomaviridae/genética , Empalme del ARN/genética , Daño del ADN/genética , Regulación Viral de la Expresión Génica/genética , Humanos , Poliadenilación/genética , Poliadenilación/fisiologíaRESUMEN
KEY MESSAGE: The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Factores de Empalme de ARN/metabolismo , Proteínas de Arabidopsis/genética , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Factores de Empalme de ARN/genéticaRESUMEN
SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155's HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155's HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform. SF3b155 residues, the mutation of which leads to cancer, contribute to the tertiary structure of the HEAT superhelix and its surface properties in the proximity of p14 and U2AF65. The molecular architecture of SF3b reveals the spatial organization of cancer-related SF3b155 mutations and advances our understanding of their effects on SF3b structure and function.
Asunto(s)
Mutación , Proteínas de Neoplasias/química , Proteínas Oncogénicas/química , Fosfoproteínas/química , Factores de Empalme de ARN/química , Empalmosomas/química , Factor de Empalme U2AF/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Genes Supresores de Tumor , Células HeLa , Humanos , Modelos Moleculares , Mariposas Nocturnas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Empalme del ARN , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Empalmosomas/metabolismo , Empalmosomas/ultraestructura , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
BACKGROUND: RBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. METHODS: In this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. RESULTS: RBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5' and 3' splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. CONCLUSIONS: RBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. GENERAL SIGNIFICANCE: The current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels.
Asunto(s)
Empalme Alternativo , Proteínas Nucleares/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Factor de Empalme U2AF/genética , Uniones Adherentes/genética , Secuencia de Aminoácidos , Arginina/metabolismo , Sitios de Unión , Daño del ADN , Regulación hacia Abajo , Endocitosis/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Perfilación de la Expresión Génica , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Aminoácido , Serina/metabolismo , Transducción de Señal , Factor de Empalme U2AF/metabolismo , Regulación hacia ArribaRESUMEN
U2 snRNP auxiliary factor 65 kDa (U2AF(65)) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF(65) stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5' splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF(65). U2AF(65) overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF(65) inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF(65) effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF(65) even inhibits general splicing of adenovirus major late (Ad ML) or ß-globin pre-mRNA. Thus, we conclude that U2AF(65) possesses a splicing Inhibitory function that leads to alternative exon skipping.
Asunto(s)
Empalme Alternativo/genética , Exones/genética , Proteínas Nucleares/genética , Ribonucleoproteínas/genética , Secuencia de Bases , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Intrones/genética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Unión Proteica , Estructura Terciaria de Proteína , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN/genética , Ribonucleoproteínas/química , Proteínas del Complejo SMN/genética , Factor de Empalme U2AF , Relación Estructura-Actividad , Factores de Transcripción/genética , Proteínas Virales/genética , Globinas beta/genética , Proteínas tau/genéticaRESUMEN
The human telomeric protein TRF1 is a component of the six-subunit protein complex shelterin, which provides telomere protection by organizing the telomere into a high-order structure. TRF1 functions as a negative regulator of telomere length by controlling the access of telomerase to telomeres. Thus, the cellular abundance of TRF1 at telomeres should be maintained and tightly regulated to ensure proper telomere function. Here, we identify U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essentialpre-mRNA splicingfactor, as a novel TRF1-interacting protein. U2AF65 interacts with TRF1 in vitro and in vivo and is capable of stabilizing TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. We also found that U2AF65 interferes with the interaction between TRF1 and Fbx4, an E3 ubiquitin ligase for TRF1. Depletion of endogenous U2AF65 expression by short interfering RNA (siRNA) reduced the stability of endogenous TRF1 whereas overexpression of U2AF65 significantly extended the half-life of TRF1. These findings demonstrate that U2AF65 plays a critical role in regulating the level of TRF1 through physical interaction and ubiquitin-mediated proteolysis. Hence, U2AF65 represents a new route for modulating TRF1 function at telomeres.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteolisis , Ribonucleoproteínas/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Ubiquitinas/metabolismo , Proteínas F-Box/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/química , Unión Proteica , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Estructura Terciaria de Proteína , Ribonucleoproteínas/química , Factor de Empalme U2AF , Proteína 1 de Unión a Repeticiones Teloméricas/química , UbiquitinaciónRESUMEN
Erythropoietic protoporphyria (EPP) results from partial deficiency of ferrochelatase (FECH). Genetically, EPP patients differ from asymptomatic mutation carriers at the unmutated FECH allele, the expression of which is modulated by single nucleotide polymorphism IVS3-48C/T. The IVS3-48C genotype, which is present among patients, leads to correct splicing of 60% of the pre-mRNA and to alternative splicing of 40%, the latter mRNA-product being destroyed by nonsense-mediated decay. An IVS3-48T genotype generates 80% correct and 20% aberrant products. Our study demonstrated that under iron deficient conditions, the aberrant splice product was increased to 56% and 50% of total FECH mRNA in erythroleukemic K562 and lymphoblastoid cell lines, respectively, both being homozygous for IVS3-48T. Concomitantly, FECH protein was decreased. Iron deficiency had less effect on the FECH splice ratio in an IVS3-48C/C lymphoblastoid cell line. Effects similar to iron deficiency were generated by siRNA knockdown of either splicing factor U2AF(65) or Fe(II)- and 2-oxoglutarate-dependent dioxygenase Jumonji domain-containing protein 6 (Jmjd6), which interacts with U2AF(65) by lysyl-hydroxylation. Based on these results, we propose that the availability of iron, a co-factor of Jmjd6, modulates U2AF(65)-lysyl-hydroxylation. This in turn, influences the relative amounts of correct and aberrant FECH mRNA splice products and thus, regulates the FECH enzyme activity.