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SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.
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Leucemia Eritroblástica Aguda/metabolismo , Mutação , Síndromes Mielodisplásicas/metabolismo , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Spliceossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Células HEK293 , Humanos , Células K562 , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Fenótipo , Fosfoproteínas/genética , Ligação Proteica , Fatores de Processamento de RNA/genética , Spliceossomos/genética , Spliceossomos/patologiaRESUMO
BACKGROUND: Small nucleolar RNAs (snoRNAs) play key roles in ribosome biosynthesis. However, the mechanism by which snoRNAs regulate cancer stemness remains to be fully elucidated. METHODS: SNORA68 expression was evaluated in breast cancer tissues by in situ hybridization and qRTâPCR. Proliferation, migration, apoptosis and stemness analyses were used to determine the role of SNORA68 in carcinogenesis and stemness maintenance. Mechanistically, RNA pull-down, RNA immunoprecipitation (RIP), cell fractionation and coimmunoprecipitation assays were conducted. RESULTS: SNORA68 exhibited high expression in triple-negative breast cancer (TNBC) and was significantly correlated with tumor size (P = 0.048), ki-67 level (P = 0.037), and TNM stage (P = 0.015). The plasma SNORA68 concentration was significantly lower in patients who achieved clinical benefit. The SNORA68-high patients had significantly shorter disease-free survival (DFS) (P = 0.036). Functionally, SNORA68 was found to promote the cell stemness and carcinogenesis of TNBC in vitro and in vivo. Furthermore, elevated SNORA68 expression led to increased nucleolar RPL23 expression and retained RPL23 in the nucleolus by binding U2AF2. RPL23 in the nucleolus subsequently upregulated c-Myc expression. This pathway was validated using a xenograft model. CONCLUSION: U2AF2-SNORA68 promotes TNBC stemness by retaining RPL23 in the nucleolus and increasing c-Myc expression, which provides new insight into the regulatory mechanism of stemness.
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Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , RNA , Núcleo Celular , Regulação Neoplásica da Expressão Gênica , Carcinogênese/genética , Proliferação de Células/genética , Fator de Processamento U2AF/genéticaRESUMO
Interleukin 7 receptor α-chain is crucial for the development and maintenance of T cells and is genetically associated with autoimmune disorders including multiple sclerosis (MS), a demyelinating disease of the CNS. Exon 6 of IL7R encodes for the transmembrane domain of the receptor and is regulated by alternative splicing: inclusion or skipping of IL7R exon 6 results in membrane-bound or soluble IL7R isoforms, respectively. We previously identified a SNP (rs6897932) in IL7R exon 6, strongly associated with MS risk and showed that the risk allele (C) increases skipping of the exon, resulting in elevated levels of sIL7R. This has important pathological consequences as elevated levels of sIL7R has been shown to exacerbate the disease in the experimental autoimmune encephalomyelitis mouse model of MS. Understanding the regulation of exon 6 splicing provides important mechanistic insights into the pathogenesis of MS. Here we report two mechanisms by which IL7R exon 6 is controlled. First, a competition between PTBP1 and U2AF2 at the polypyrimidine tract (PPT) of intron 5, and second, an unexpected U2AF2-mediated assembly of spicing factors in the exon. We noted the presence of a branchpoint sequence (BPS) (TACTAAT or TACTAAC) within exon 6, which is stronger with the C allele. We also noted that the BPS is followed by a PPT and conjectured that silencing could be mediated by the binding of U2AF2 to that tract. In support of this model, we show that evolutionary conservation of the exonic PPT correlates well with the degree of alternative splicing of exon 6 in two non-human primate species and that U2AF2 binding to this PPT recruits U2 snRNP components to the exon. These observations provide the first explanation for the stronger silencing of IL7R exon 6 with the disease associated C allele at rs6897932.
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Variants in the RNA binding protein (RBP) U2AF2 are hypothesized to cause a novel neurodevelopmental disorder. Here, we report a patient with a de novo missense variant in U2AF2, the second case report of the same variant, and third case report overall. The patient in this report has a history of global developmental delay, dysmorphic features, and epilepsy. This presentation is consistent with the previous case report with the same U2AF2 variant and with a recent case report of another U2AF2 variant, strengthening the evidence that variants in U2AF2 are the cause of a novel neurodevelopmental disorder.
Assuntos
Epilepsia , Deficiência Intelectual , Anormalidades Musculoesqueléticas , Transtornos do Neurodesenvolvimento , Criança , Humanos , Deficiências do Desenvolvimento/genética , Transtornos do Neurodesenvolvimento/genética , Mutação de Sentido Incorreto/genética , Epilepsia/diagnóstico , Epilepsia/genética , Deficiência Intelectual/genética , Fator de Processamento U2AF/genéticaRESUMO
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.
Assuntos
Mutação de Sentido Incorreto , Proteínas de Neoplasias , Neoplasias , Splicing de RNA , RNA Neoplásico , Fator de Processamento U2AF , Motivos de Aminoácidos , Substituição de Aminoácidos , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/química , Neoplasias/genética , Neoplasias/metabolismo , RNA Neoplásico/química , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Fator de Processamento U2AF/química , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismoRESUMO
The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF65 and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.
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Processamento Alternativo , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Spliceossomos/genética , Fator de Processamento U2AF/genética , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , DNA Complementar/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Motivos de Nucleotídeos , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/metabolismo , Fator de Processamento U2AF/antagonistas & inibidores , Fator de Processamento U2AF/metabolismoRESUMO
Background LINC00665 is a newly identified oncogene, which has been reported to be oncogene in various cancers. Nevertheless, its role in the progression of colorectal cancer (CRC) remains obscure to the extent. This study aimed at exploring the role and mechanism of LINC00665 in CRC progression. Materials and methods RNA and protein expression were detected via qRT-PCR and western blot. Functional assays were conducted to investigate the role of LINC00665 in the CRC cellular processes. TOP/FOP assay was performed to detect the activity of Wnt/ß-catenin signaling pathway. Mechanism investigations were carried out to explore the regulatory relationship among genes. Results LINC00665 was overtly expressed in CRC cell lines at high levels. Functionally, silencing of LINC00665 could curb in vitro CRC cell growth, migration and invasion, while stimulating cell apoptosis. Mechanically, LINC00665 sponged miR-214-3p to up-regulate CTNNB1 expression, consequently activating Wnt/ß-catenin signaling pathway. Furthermore, LINC00665 could bind to U2AF2 and enhance the association between U2AF2 and CTNNB1, increasing the stability of CTNNB1. CTNNB1 overexpression could reverse the suppressive effects of LINC00665 downregulation. Conclusion LINC00665 stimulates CRC progression through the activation of Wnt/ß-catenin signaling pathway, which hopefully might be a therapeutic target for CRC.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , Proteína Wnt1/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genéticaRESUMO
Lung adenocarcinoma (LUAD) is difficult to cureradically. Long non-coding RNAs (lncRNAs) in LUAD are a hotspot in molecular research, however, the role of lncRNA ASMTL-AS1 in LUAD is still unknown. Our study explores the role and mechanisms of ASMTL-AS1 in LUAD. Quantitative reverse transcription PCR or western blot was utilized to analyze the expression of RNAs or proteins. The influences of ASMTL-AS1 and SAT1 on LUAD cells were analyzed by functional assays. Biological instruments were applied to observe ferroptosis-related markers. In vivo assays were performed to uncover the impact of ASMTL-AS1 on LUAD. Moreover, mechanism assays were done to confirm the relationship among ASMTL-AS1, SAT1 and U2AF2. Results showed that ASMTL-AS1 was down-regulated in LUAD cells and ASMTL-AS1 up-regulation resulted in retarded LUAD cell and xenograft tumor growth along with stimulated ferroptosis. ASMTL-AS1 recruited U2AF2 to stabilize SAT1 mRNA. Furthermore, SAT1 exerted a cancer suppressor role in LUAD cells. In conclusion, we first demonstrated that ASMTL-AS1 positively regulated SAT1 to promote ferroptosis and could stabilize SAT1 mRNA via recruiting U2AF2, shedding a light on a novel molecular mechanism in LUAD progression.
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Acetiltransferases/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Ferroptose , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metiltransferases/metabolismo , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Pre-mRNA splicing is a key post-transcriptional regulation process in which introns are excised and exons are ligated together. A novel class of structured intron was recently discovered in fish. Simple expansions of complementary AC and GT dimers at opposite boundaries of an intron were found to form a bridging structure, thereby enforcing correct splice site pairing across the intron. In some fish introns, the RNA structures are strong enough to bypass the need of regulatory protein factors for splicing. Here, we discuss the prevalence and potential functions of highly structured introns. In humans, structured introns usually arise through the co-occurrence of C and G-rich repeats at intron boundaries. We explore the potentially instructive example of the HLA receptor genes. In HLA pre-mRNA, structured introns flank the exons that encode the highly polymorphic ß sheet cleft, making the processing of the transcript robust to variants that disrupt splicing factor binding. While selective forces that have shaped HLA receptor are fairly atypical, numerous other highly polymorphic genes that encode receptors contain structured introns. Finally, we discuss how the elevated mutation rate associated with the simple repeats that often compose structured intron can make structured introns themselves rapidly evolving elements.
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Conformação de Ácido Nucleico , Splicing de RNA , RNA/química , RNA/genética , Animais , Evolução Biológica , Éxons , Humanos , Íntrons , Polimorfismo de Nucleotídeo Único , Precursores de RNA/química , Precursores de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Fator de Processamento U2AF/metabolismo , Relação Estrutura-AtividadeRESUMO
Intervertebral disc degeneration (IDD) is one of the major leading causes of back pain affecting the patient's quality of life. However, the roles of circular RNA (circRNA) in IDD remains unclear. This study aimed to explore the function and underlying mechanism of circ_0036763 in IDD. In this study, expressions of circ_0036763, U2 small nuclear RNA auxiliary factor 2 (U2AF2), miR-583 and aggrecan (ACAN) in primary human nucleus pulposus cells (HNPCs) derived from IDD patients and healthy controls were detected by quantitative real-time reverse transcription-PCR (qRT-PCR) or Western blot (WB). The relationship between pre-circ_0036763 and U2AF2, circ_0036763 and miR-583, miR-583 and ACAN mRNA was determined by bioinformatic analysis, miRNA pull down or RNA immunoprecipitation (RIP) assay. The expressions of Collagen I and Collagen II were evaluated by WB. Co-culture of bone marrow mesenchymal stem cells (bMSCs) or bMSCs-derived exosomes and HNPCs were performed to identify the effect of U2AF2 on the mature of circ_0036763 and ACAN. Results indicated that circ_0036763, U2AF2 and ACAN were downregulated while miR-583 was upregulated in HNPCs derived from IDD patients compared with that in normal HNPCs. Besides, overexpression of circ_0036763 elevated the expressions of ACAN and Collagen II whereas reduced Collagen I expression in HNPCs. Moreover, U2AF2 promoted the mature of circ_0036763, and circ_0036763 positively regulated ACAN by directly sponging miR-583. Furthermore, exosomal U2AF2 derived from bMSCs could increase U2AF2 levels in HNPCs and subsequently regulate the expression of ACAN by circ_0036763/miR-583 axis. In summary, circ_0036763 modified by exosomal U2AF2 derived from bMSCs alleviated IDD through regulating miR-583/ACAN axis in HNPCs. Thus, this study might provide novel therapeutic targets for IDD.
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Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3' untranslated region (3'UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3'UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3'UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3' UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.
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Vírus da Encefalite Japonesa (Espécie) , Flavivirus , Doenças dos Suínos , Infecção por Zika virus , Zika virus , Humanos , Animais , Suínos , Flavivirus/genética , Regiões 3' não Traduzidas , Ribonucleoproteína Nuclear Pequena U2/genética , Infecção por Zika virus/genética , Infecção por Zika virus/veterinária , Replicação Viral/genética , Linhagem Celular , Zika virus/genética , Zika virus/metabolismo , Vírus da Encefalite Japonesa (Espécie)/genética , RNA Viral/genética , RNA Viral/metabolismo , Fator de Processamento U2AF/genética , Doenças dos Suínos/genéticaRESUMO
Mutations in RNA splicing factor genes including SF3B1, U2AF1, SRSF2, and ZRSR2 have been reported to contribute to development of myeloid neoplasms including myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Chemical tools targeting cells carrying these mutant genes remain limited and underdeveloped. Among the four proteins, mutant U2AF1 (U2AF1mut) acquires an altered 3' splice site selection preference and co-operates with the wild-type U2AF1 (U2AF1wt) to change various gene isoform patterns to support MDS cells survival and proliferation. U2AF1 mutations in MDS cells are always heterozygous and the cell viability is reduced when exposed to additional insult affecting U2AF1wt function. To investigate if the pharmacological inhibition of U2AF1wt function can provoke drug-induced vulnerability of cells harboring U2AF1 mut , we conducted a fragment-based library screening campaign to discover compounds targeting the U2AF homology domain (UHM) in U2AF1 that is required for the formation of the U2AF1/U2AF2 complex to define the 3' splice site. The most promising hit (SF1-8) selectively inhibited growth of leukemia cell lines overexpressingU2AF1 mut and human primary MDS cells carrying U2AF1 mut . RNA-seq analysis of K562-U2AF1mut following treatment with SF1-8 further revealed alteration of isoform patterns for a set of proteins that impair or rescue pathways associated with endocytosis, intracellular vesicle transport, and secretion. Our data suggested that further optimization of SF1-8 is warranted to obtain chemical probes that can be used to evaluate the therapeutic concept of inducing lethality to U2AF1 mut cells by inhibiting the U2AF1wt protein.
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Objective: As an important factor tumor regulatorï¼long non-coding RNAs (lncRNAs) have aroused extensive attention via the diverse functional mechanisms that were associated with the pathological and physiological processes of HCC. Here, the main purpose of this study was to provide a clear understanding about the expression, functions and potential mechanism of lncRNA CECR7 (Cat Eye Syndrome Chromosome Region, Candidate 7) in HCC. Methods: RT-qPCR analysis and TCGA database analysis were applied to investigate the expression of CECR7 in HCC cell lines and tissues. Chi-squared Test was employed to explore the correlation between CECR7 expression and HCC clinicopathological features. Besides, Kaplan-Meier curves were constructed to test the effects of CECR7 expression on the prognosis of HCC patients. Transwell assays, MTT assay EdU assay and animal experiments were applied to explore the effects of CECR7 expression on HCC cells migration, invasion, and growth. Furthermore, RNA-seq analysis, luciferase reporter assay and mRNA decay rates assessment were utilized to investigate the mechanism whereby CECR7 regulated EXO1 mRNA. And, rescue experiments were used to determine whether EXO1 was an essential mediator for CECR7 to accelerate HCC cells migration, invasion, and growth. Results: CECR7 was determined to be significantly overexpressed in HCC cell lines and tissues. CECR7 expression was closely correlated with the tumor size, venous infiltration, TNM stage, 5-year overall survival and disease-free survival of HCC. And, CECR7 played a catalytic role in HCC cells migration, invasion, and growth. Furthermore, CECR7 enhanced the stability of EXO1 mRNA by recruiting RNA binding protein U2AF2. And, EXO1 was determined to be an essential mediator for CECR7 to accelerate HCC cells migration, invasion, and growth. Conclusion: In a word, our findings demonstrates that the cancer-promoting gene lncRNA CECR7 motivates HCC metastasis and growth through enhanced mRNA stability of EXO1 mediated by U2AF2, proposing a new insight for targeted therapy of HCC.
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BACKGROUND: Ischaemic stroke is the leading cause of mortality and disability in the world. LncRNA NEAT1 has been shown to play an important role in ischaemic injury, but the molecular mechanism remains unclear. METHODS: qRT-PCR was used to determine the expression of lncRNA NEAT1 in OGD/R-induced BV-2 cells. Cell viability was assessed by an MTT assay, and cell apoptosis was assessed by flow cytometry. The expression of related proteins was evaluated by Western blotting and ELISA. The interactions among lncRNA NEAT1, U2AF2 and Wnt3a mRNA was demonstrated by RIP and RNA pulldown assays. XAV-939 was used as an inhibitor of the Wnt/ß-catenin pathway. RESULTS: LncRNA NEAT1 was found to be downregulated in OGD/R-induced BV-2 cells. Overexpression of lncRNA NEAT1 protected BV-2 cells against OGD/R-induced injury. LncRNA NEAT1 enhanced the stability of Wnt3a mRNA via U2AF2. Knockdown of Wnt3a or blockade of the Wnt/ß-catenin pathway rescued the effect of lncRNA NEAT1. CONCLUSIONS: LncRNA NEAT1 protected cells against OGD/R-induced apoptosis and the inflammatory response by activating the Wnt/ß-catenin pathway through upregulation of Wnt3a in a U2AF2-dependent manner. LncRNA NEAT1 could be a promising therapeutic candidate for ischaemic stroke treatment in the future.
Assuntos
Isquemia Encefálica , AVC Isquêmico , MicroRNAs , RNA Longo não Codificante , Via de Sinalização Wnt , Proteína Wnt3A , beta Catenina , Apoptose/genética , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Humanos , AVC Isquêmico/genética , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro , Fator de Processamento U2AF/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The dysregulation of U2 Small Nuclear RNA Auxiliary Factor 2 (U2AF2) is associated with malignant behaviors of multiple types of tumors. In this study, we explored the association between U2AF2 dysregulation and the survival of patients with primary leiomyosarcoma, the regulatory effect of U2AF2 on cell growth/aerobic glycolysis, and the mechanisms of U2AF2 dysregulation at the transcriptional level. Gene expression and survival time of patients with primary leiomyosarcoma were extracted from TCGA-Sarcoma (SARC). Leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1 were utilized to construct in vitro and in vivo models. Results showed that the higher U2AF2 expression group had significantly shorter progression-free survival (HR: 2.049, 95%CI: 1.136-3.697, p = 0.011) and disease-specific survival (4.656, 95%CI: 2.141-10.13, p < 0.001) compared to the lower U2AF2 expression group. U2AF2 knockdown suppressed leiomyosarcoma cell growth and aerobic glycolysis (decreased glucose uptake, lactate production, and extracellular acidification rate) in vitro. Tumors derived from SK-LMS-1 cells with U2AF2 knockdown grew significantly slower, with lower GLUT1, PGK1, and PGAM1 protein expression than the control groups. TFDP1 and E2F1 could interact with each other in leiomyosarcoma cells. Both TFDP1 and E2F1 could bind to the promoter of U2AF2 and exert a synergistic activating effect on U2AF2 transcription. In conclusion, this study revealed that U2AF2 upregulation is associated with poor survival of leiomyosarcoma. Its upregulation enhances proliferation and aerobic glycolysis of leiomyosarcoma cells in vitro and in vivo. TFDP1 and E2F1 can form a complex, which binds to the U2AF2 gene promoter and synergistically activates its transcription.
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Leiomiossarcoma , Fatores de Transcrição E2F/metabolismo , Glicólise/genética , Humanos , Leiomiossarcoma/genética , RNA Nuclear Pequeno , Fator de Processamento U2AF , Fator de Transcrição DP1/genética , Fator de Transcrição DP1/metabolismoRESUMO
Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.
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Neoplasias/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Fator de Processamento U2AF/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Programas de Rastreamento , Simulação de Dinâmica Molecular , Mutação/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Splicing de RNA/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Spliceossomos/efeitos dos fármacos , Interface Usuário-ComputadorRESUMO
BACKGROUND: 1q21.3 amplification, which is frequently observed in metastatic melanoma, is associated with cancer progression. Interleukin enhancer-binding factor 2 (ILF2) is located in the 1q21.3 amplified region, but its functional role or contribution to tumour aggressiveness in cutaneous melanoma is unknown. METHODS: In silico analyses were performed using the TCGA SKCM dataset with clinical annotations and three melanoma microarray cohorts from the GEO datasets. RNA in situ hybridisation and immunohistochemistry were utilised to validate the gene expression in melanoma tissues. Four stable melanoma cell lines were established for in vitro ILF2 functional characterisation. RESULTS: Our results showed that the ILF2 copy number variation (CNV) is positively correlated with ILF2 mRNA expression (r = 0.68, p < .0001). Additionally, ILF2 expression is significantly increased with melanoma progression (p < .0001), and significantly associated with poor overall survival for metastatic melanoma patients (p = .026). The overexpression of ILF2 (ILF2-OV) promotes proliferation in metastatic melanoma cells, whereas ILF2 knockdown decreases proliferation by blocking the cell cycle. Mechanistically, we demonstrated the interaction between ILF2 and the splicing factor U2AF2, whose knockdown reverses the proliferation effects mediated by ILF2-OV. Stage IIIB-C melanoma patients with high ILF2-U2AF2 expression showed significantly shorter overall survival (p = .024). Enhanced ILF2/U2AF2 expression promotes a more efficient DNA-damage repair by increasing RAD50 and ATM mRNA expression. Paradoxically, metastatic melanoma cells with ILF2-OV were more sensitive to ATM inhibitors. CONCLUSION: Our study uncovered that ILF2 amplification of the 1q21.3 chromosome is associated with melanoma progression and triggers a functional downstream pathway in metastatic melanoma promoting drug resistance.
Assuntos
Proliferação de Células/genética , Dano ao DNA/genética , Melanoma/genética , Proteína do Fator Nuclear 45/genética , Proteína do Fator Nuclear 45/metabolismo , Neoplasias Cutâneas/genética , Linhagem Celular Tumoral , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Humanos , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND: Glioma is the most common and lethal primary brain tumor in adults, and angiogenesis is one of the key factors contributing to its proliferation, aggressiveness, and malignant transformation. However, the discovery of novel oncogenes and the study of its molecular regulating mechanism based on circular RNAs (circRNAs) may provide a promising treatment target in glioma. METHODS: Bioinformatics analysis, qPCR, western blotting, and immunohistochemistry were used to detect the expression levels of ISL2, miR-342-3p, circRNA ARF1 (cARF1), U2AF2, and VEGFA. Patient-derived glioma stem cells (GSCs) were established for the molecular experiments. Lentiviral-based infection was used to regulate the expression of these molecules in GSCs. The MTS, EDU, Transwell, and tube formation assays were used to detect the proliferation, invasion, and angiogenesis of human brain microvessel endothelial cells (hBMECs). RNA-binding protein immunoprecipitation, RNA pull-down, dual-luciferase reporter, and chromatin immunoprecipitation assays were used to detect the direct regulation mechanisms among these molecules. RESULTS: We first identified a novel transcription factor related to neural development. ISL2 was overexpressed in glioma and correlated with poor patient survival. ISL2 transcriptionally regulated VEGFA expression in GSCs and promoted the proliferation, invasion, and angiogenesis of hBMECs via VEGFA-mediated ERK signaling. Regarding its mechanism of action, cARF1 upregulated ISL2 expression in GSCs via miR-342-3p sponging. Furthermore, U2AF2 bound to and promoted the stability and expression of cARF1, while ISL2 induced the expression of U2AF2, which formed a feedback loop in GSCs. We also showed that both U2AF2 and cARF1 had an oncogenic effect, were overexpressed in glioma, and correlated with poor patient survival. CONCLUSIONS: Our study identified a novel feedback loop among U2AF2, cARF1, miR-342-3p, and ISL2 in GSCs. This feedback loop promoted glioma angiogenesis, and could provide an effective biomarker for glioma diagnosis and prognostic evaluation, as well as possibly being used for targeted therapy.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Glioma/patologia , Proteínas com Homeodomínio LIM/metabolismo , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/patologia , Proteínas do Tecido Nervoso/metabolismo , RNA Circular/genética , Fator de Processamento U2AF/metabolismo , Fatores de Transcrição/metabolismo , Fator 1 de Ribosilação do ADP/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Humanos , Proteínas com Homeodomínio LIM/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas do Tecido Nervoso/genética , Prognóstico , Fator de Processamento U2AF/genética , Taxa de Sobrevida , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3' splice sites (3'ss). Both proteins preferentially bind uridine-rich sequences upstream of 3'ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3'ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3'ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3'ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.
RESUMO
Increasing evidence has confirmed that deubiquitinating enzymes play an important role in lung cancer progression. In the current study, we investigated the expression profile of deubiquitinating enzymes in non-small cell lung cancer (NSCLC) tissues and identified OTUB2 as an upregulated deubiquitinating enzyme. The role of OTUB2 in NSCLC is unknown. Methods: Quantitative, real-time PCR and Western blot were used to detect OTUB2 and U2AF2 expression in NSCLC tissues. The correlations between OTUB2 and U2AF2 expression and clinicopathologic features were then analyzed. We used In vitro Cell Counting Kit-8 (CCK-8) , colony formation , and trans-well invasion assays to investigate the function of OTUB2 and U2AF2 in tumorigenesis. The regulation of glycolysis by OTUB2 and U2AF2 was assessed by determining the extracellular acid ratio, glucose consumption, and lactate production. The mechanism of OTUB2 was explored through co-immunoprecipitation and mass spectrometry analyses. A xenograft model was also used to study the tumorigenesis role of OTUB2 In vivo. Results: OTUB2 expression was significantly upregulated in primary NSCLC tissues and greatly associated with metastasis, advanced tumor stages, poor survival, and recurrence. In NSCLC cell lines, OTUB2 promoted cell growth, colony formation, migration, and invasive activities. Mechanistic investigations showed that OTUB2 stimulated the Warburg effect and induced the activation of the serine/threonine kinase/mechanistic target of rapamycin kinase (AKT/mTOR) pathway in different NSCLC cells. More importantly, OTUB2 promoted NSCLC progression, which was largely dependent on the direct binding to and deubiquitination of U2AF2, at least in NSCLC cells. U2AF2 expression was also significantly upregulated in primary NSCLC tissues and dramatically associated with metastasis, advanced tumor stages, poor survival, and recurrence. Importantly, a positive correlation between the protein expression of OTUB2 and U2AF2 in NSCLC tissues was found. In vivo experiments indicated that OTUB2 promoted xenograft tumor growth of NSCLC cell. In addition, our results suggest that high expression of OTUB2, U2AF2 and PGK1 is significantly associated with worse prognosis in NSCLC patients. Conclusion: Taken together, the present study provides the first evidence that OTUB2 acts as a pivotal driver in NSCLC tumorigenesis by stabilizing U2AF2 and activating the AKT/mTOR pathway and the Warburg effect. It may serve as a new potential prognostic indicator and therapeutic target in NSCLC.