RESUMO
Aberrant alternative splicing events play a critical role in cancer biology, contributing to tumor invasion, metastasis, epithelial-mesenchymal transition, and drug resistance. Recent studies have shown that alternative splicing is a key feature for transcriptomic variations in colorectal cancer, which ranks third among malignant tumors worldwide in both incidence and mortality. Long non-coding RNAs can modulate this process by acting as trans-regulatory agents, recruiting splicing factors, or driving them to specific targeted genes. LncH19 is a lncRNA dis-regulated in several tumor types and, in colorectal cancer, it plays a critical role in tumor onset, progression, and metastasis. In this paper, we found, that in colorectal cancer cells, the long non-coding RNA H19 can bind immature RNAs and splicing factors as hnRNPM and RBFOX2. Through bioinformatic analysis, we identified 57 transcripts associated with lncH19 and containing binding sites for both splicing factors, hnRNPM, and RBFOX2. Among these transcripts, we identified the mRNA of the GTPase-RAC1, whose alternatively spliced isoform, RAC1B, has been ascribed several roles in the malignant transformation. We confirmed, in vitro, the binding of the splicing factors to both the transcripts RAC1 and lncH19. Loss and gain of expression experiments in two colorectal cancer cell lines (SW620 and HCT116) demonstrated that lncH19 is required for RAC1B expression and, through RAC1B, it induces c-Myc and Cyclin-D increase. In vivo, investigation from biopsies of colorectal cancer patients showed higher levels of all the explored genes (lncH19, RAC1B, c-Myc and Cyclin-D) concerning the healthy counterpart, thus supporting our in vitro model. In addition, we identified a positive correlation between lncH19 and RAC1B in colorectal cancer patients. Finally, we demonstrated that lncH19, as a shuttle, drives the splicing factors RBFOX2 and hnRNPM to RAC1 allowing exon retention and RAC1B expression. The data shown in this paper represent the first evidence of a new mechanism of action by which lncH19 carries out its functions as an oncogene by prompting colorectal cancer through the modulation of alternative splicing.
Assuntos
Processamento Alternativo , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Fatores de Processamento de RNA , RNA Longo não Codificante , Proteínas rac1 de Ligação ao GTP , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , RNA Longo não Codificante/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Linhagem Celular Tumoral , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND: The identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4. METHODS: In vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4's interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations. RESULTS: PARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4's tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss. CONCLUSIONS: PARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex-unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Neoplasias Pulmonares , Proteínas Nucleares , Animais , Humanos , Camundongos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Ligação Proteica , Splicing de RNA , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Íntrons , Elementos Nucleotídeos Longos e Dispersos , Splicing de RNA , RNA de Cadeia Dupla , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Interferons/metabolismo , Interferons/genética , Animais , Células HEK293 , Camundongos , Transcriptoma , Éxons , Sítios de Splice de RNA , Elementos Alu/genéticaRESUMO
Coronary atherosclerosis-induced myocardial ischemia leads to cardiomyocyte apoptosis. The regulatory mechanisms for cardiomyocyte apoptosis have not been fully understood. Circular RNAs are non-coding RNAs which play important roles in heart function maintenance and progression of heart diseases by regulating gene transcription and protein translation. Here, we reported a conserved cardiac circular RNA, which is generated from the second exon of LRP6 and named circLRP62-2 . CircLRP62-2 can protect cardiomyocyte from hypoxia-induced apoptosis. The expression of circLRP62-2 in cardiomyocytes was down-regulated under hypoxia, while forced expression of circLRP62-2 inhibited cell apoptosis. Normally, circLRP62-2 was mainly localized in the nucleus. Under hypoxia, circLRP62-2 is associated with heterogeneous nuclear ribonucleoprotein M (hnRNPM) to be translocated into the cytoplasm. It recruited hnRNPM to fibroblast growth factor 9 (FGF9) mRNA to enhance the expression of FGF9 protein, promoting hypoxia-adaption and viability of cardiomyocytes. In summary, this study uncovers a new inhibitor of apoptosis and reveals a novel anti-apoptotic pathway composed of circLRP62-2 , hnRNPM, and FGF9, which may provide therapeutic targets for coronary heart disease and ischemic myocardial injury.
Assuntos
MicroRNAs , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , RNA Circular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Fator 9 de Crescimento de Fibroblastos/metabolismo , Apoptose/genética , Hipóxia/metabolismo , MicroRNAs/genéticaRESUMO
BACKGROUND & AIMS: Cancer stemness and immune evasion are closely associated and play critical roles in tumor development and resistance to immunotherapy. However, little is known about the underlying molecular mechanisms that coordinate this association. METHODS: The expressions of heterogeneous nuclear ribonucleoprotein M (HNRNPM) in 240 hepatocellular carcinoma (HCC) samples, public databases, and liver development databases were analyzed. Chromatin immunoprecipitation assays were performed to explore the associations between stem-cell transcription factors and HNRNPM. HNRNPM-regulated alternative splicing (AS) and its binding motif were identified by RNA-seq and RIP-seq. HNRNPM-specific antisense oligonucleotides were developed to explore potential therapeutic targets in HCC. CD8+ T cells that were co-cultured with tumor cells were sorted by flow cytometry assays. RESULTS: We identified an elevated oncofetal splicing factor in HCC, HNRNPM, that unifies and regulates the positive association between cancer stemness and immune evasion. HNRNPM knockdown abolished HCC tumorigenesis and diminished cancer stem cell properties in vitro and in vivo. Mechanistically, HNRNPM regulated the AS of MBD2 by binding its flanking introns, whose isoforms played opposing roles. Although MBD2a and MBD2c competitively bound to CpG islands in the FZD3 promoter, MBD2a preferentially increased FZD3 expression and then activated the WNT/ß-catenin pathway. Interestingly, FZD3 and ß-catenin further provided additional regulation by targeting OCT4 and SOX2. We found that HNRNPM inhibition significantly promoted CD8+ T cell activation and that HNRNPM- antisense oligonucleotides effectively inhibited WNT/ß-catenin to enhance anti-programmed cell death protein-1 immunotherapy by promoting CD8+ T cell infiltration. CONCLUSIONS: HNRNPM has a tumor-intrinsic function in generating an immunosuppressive HCC environment through an AS-dependent mechanism and demonstrates proof of the concept of targeting HNRNPM in tailoring HCC immunotherapeutic approaches.
Assuntos
Carcinoma Hepatocelular , Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Neoplasias Hepáticas , Carcinoma Hepatocelular/patologia , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Oligonucleotídeos Antissenso , beta Catenina/metabolismoRESUMO
Circular RNAs (circRNAs) have emerged as key regulators of human cancers, yet their modes of action in gastric cancer (GC) remain largely unknown. Here, we identified circURI1 back-spliced from exons 3 and 4 of unconventional prefoldin RPB5 interactor 1 (URI1) from circRNA profiling of five-paired human gastric and the corresponding nontumor adjacent specimens (paraGC). CircURI1 exhibits the significantly higher expression in GC compared with paraGC and inhibitory effects on cell migration and invasion in vitro and GC metastasis in vivo. Mechanistically, circURI1 directly interacts with heterogeneous nuclear ribonucleoprotein M (hnRNPM) to modulate alternative splicing of genes, involved in the process of cell migration, thus suppressing GC metastasis. Collectively, our study expands the current knowledge regarding the molecular mechanism of circRNA-mediated cancer metastasis via modulating alternative splicing.
Assuntos
Processamento Alternativo/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Metástase Neoplásica/genética , RNA Circular/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Proliferação de Células , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Experimentais , RNA Circular/genéticaRESUMO
High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.
Assuntos
Adenocarcinoma/metabolismo , Proliferação de Células , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Neoplasias da Próstata/metabolismo , Splicing de RNA , RNA Circular/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Masculino , Camundongos SCID , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Circular/genética , Carga Tumoral , Células Tumorais CultivadasRESUMO
CDR1as is a well-identified circular RNA with regulatory roles in a variety of physiological processes. However, the effects of CDR1as on stemness of periodontal ligament stem cells (PDLSCs) and the underlying mechanisms remain unclear. In this study, we detect CDR1as in human PDLSCs, and subsequently demonstrate that CDR1as maintains PDLSC stemness. Knockdown of CDR1as decreases the expression levels of stemness-related genes and impairs the cell's multi-differentiation and cell migration abilities, while overexpression of CDR1as increases the expression levels of stemness-related genes and enhances these abilities. Furthermore, our results indicate that the RNA-binding protein hnRNPM directly interacts with CDR1as and regulates its expression in PDLSCs. In addition, we show that CDR1as promotes the expression of stemness-related genes in PDLSCs by inhibiting miR-7-mediated suppression of KLF4 expression. Collectively, our results demonstrate that CDR1as participates in the molecular circuitry that regulates PDLSC stemness.
Assuntos
Autoantígenos/metabolismo , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/genética , Proteínas do Tecido Nervoso/metabolismo , Ligamento Periodontal/citologia , Células-Tronco/citologia , Adolescente , Adulto , Apoptose , Autoantígenos/genética , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Ligamento Periodontal/metabolismo , Células-Tronco/metabolismo , Adulto JovemRESUMO
RNA metabolism involves complex and regulated processes, some of which include transcription, intracellular transport, translation, and degradation. The involvement of RNA binding proteins in these processes remains mostly uncharacterized regarding brain functions, especially cognition. In this study, we report that knockdown of hnRNPM in the CA1 hippocampal region of the mouse brain leads to learning and memory impairment. This finding is further supported, by the reduction of pre- and post-synaptic protein levels synaptophysin and PSD95. Notably, loss of hnRNPM affects the physiological spine in vivo by impairing the morphology of the dendritic spines. Additionally, our study demonstrates that hnRNPM directly binds to the 3'UTR of synaptophysin and PSD95 mRNAs, resulting in the stabilization of these mRNAs. Together, these findings present novel insight into the regulatory role of hnRNPM in neuronal structure and function.
Assuntos
Disfunção Cognitiva/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Plasticidade Neuronal , Animais , Região CA1 Hipocampal/crescimento & desenvolvimento , Região CA1 Hipocampal/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Disfunção Cognitiva/genética , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/deficiência , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade de RNA , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMO
One compound sometimes shows two biological functions, becoming important aspect of recent drug discovery. This study began with an attempt to confirm the previously reported molecular mechanism of the anti-human immunodeficiency virus (HIV) heterocyclic compound BMMP [2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine], i.e., induction of abnormal uncoating of the viral core at the post-entry step. Our mechanistic study gave results consistent with this mechanism. We further attempted to find out the molecular target of BMMP by a pulldown approach using previously synthesized biotinylated BMMP (Biotin-BMMP) and successfully identified heterogenous nuclear ribonucleoprotein M (hnRNP M) as a BMMP-binding protein. This protein was found not to be accountable for the anti-HIV activity of BMMP. As hnRNP M has been reported to promote cancer metastasis, we tested this mechanism and found that BMMP suppressed migration of the human lung carcinoma cell line A549 stimulated with transforming growth factor-ß (TGF-ß). Mechanistic study showed that BMMP suppressed the expression of CD44 mRNA via the regulation of hnRNP M. Furthermore, six new derivatives of BMMP were synthesized, and the patterns of their activities against HIV-1 and cell migration were not uniform, suggesting that the anti-HIV mechanism and the anti-cell migration mechanism of BMMP are independent. Taken together, the anti-cell migration activity of the anti-HIV heterocyclic compound BMMP was newly discovered by identification of its binding protein hnRNP M using a chemical biology approach.
Assuntos
Fármacos Anti-HIV/química , Compostos Heterocíclicos/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Compostos Heterocíclicos/metabolismo , Compostos Heterocíclicos/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Ligação Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismoRESUMO
Aim: This study aimed to investigate the expression of microRNA-505 (miR-505) and explore its clinical significance, biological function and mechanisms in hepatocellular carcinoma (HCC). Methods: Expression of miR-505 was measured in 128 paired HCC tissues and five cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). MTT assay, Transwell migration, invasion assays and apoptosis assay were performed to explore the functional role of miR-505. The target gene of miR-505 was assessed using the bioinformatics assay and the related signaling pathway was confirmed using western blot. Results: Expression of miR-505 in HCC serum and tissues were downregulated. The overexpression of miR-505 in HCC cells inhibited cell proliferation and metastasis, as well as enhanced cell apoptosis by directly downregulating heterogeneous nuclear ribonucleoprotein M (HNRNPM). The activity of the Wnt/ß-catenin signaling pathway was suppressed by the overexpression of miR-505 but was promoted by the upregulation of HNRNPM. Conclusion: The results suggest that the regulation of miR-505/HNRNPM may be a novel strategy to improve the targeted therapy of HCC.
Assuntos
Apoptose/genética , Carcinoma Hepatocelular/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Via de Sinalização Wnt/genética , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Neoplasias Hepáticas/genéticaRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. While pathogenic mutations in the DNA/RNA-binding protein Matrin-3 (MATR3) are linked to ALS and distal myopathy, the molecular mechanisms underlying MATR3-mediated neuromuscular degeneration remain unclear. METHODS: We generated Drosophila lines with transgenic insertion of human MATR3 wildtype, disease-associated variants F115C and S85C, and deletion variants in functional domains, ΔRRM1, ΔRRM2, ΔZNF1 and ΔZNF2. We utilized genetic, behavioral and biochemical tools for comprehensive characterization of our models in vivo and in vitro. Additionally, we employed in silico approaches to find transcriptomic targets of MATR3 and hnRNPM from publicly available eCLIP datasets. RESULTS: We found that targeted expression of MATR3 in Drosophila muscles or motor neurons shorten lifespan and produces progressive motor defects, muscle degeneration and atrophy. Strikingly, deletion of its RNA-recognition motif (RRM2) mitigates MATR3 toxicity. We identified rump, the Drosophila homolog of human RNA-binding protein hnRNPM, as a modifier of mutant MATR3 toxicity in vivo. Interestingly, hnRNPM physically and functionally interacts with MATR3 in an RNA-dependent manner in mammalian cells. Furthermore, common RNA targets of MATR3 and hnRNPM converge in biological processes important for neuronal health and survival. CONCLUSIONS: We propose a model of MATR3-mediated neuromuscular degeneration governed by its RNA-binding domains and modulated by interaction with splicing factor hnRNPM.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Degeneração Neural/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Camundongos , Degeneração Neural/patologia , Motivos de Ligação ao RNA/fisiologiaRESUMO
Alternative splicing has been shown to causally contribute to the epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein-protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Processamento Alternativo/genética , Transição Epitelial-Mesenquimal/genética , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Células HCT116 , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Domínios e Motivos de Interação entre Proteínas , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein's ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Imunidade Inata/genética , Interleucina-6/metabolismo , Macrófagos/imunologia , Splicing de RNA/imunologia , Processamento Alternativo/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Éxons , Expressão Gênica , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Ontologia Genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/imunologia , Humanos , Interleucina-6/genética , Íntrons , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/virologia , Camundongos , Mutação , Fosforilação , Células RAW 264.7 , Splicing de RNA/genética , RNA-Seq , Salmonella/fisiologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and MDA5, initiates innate antiviral responses. Although regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we identified heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a negative regulator of RLR-mediated signaling. Overexpression of hnRNPM markedly inhibited RNA virus-triggered innate immune responses. Conversely, hnRNPM-deficiency increased viral RNA-triggered innate immune responses and inhibited replication of RNA viruses. Viral infection caused translocation of hnRNPM from the nucleus to the cytoplasm. hnRNPM interacted with RIG-I and MDA5, and impaired the binding of the RLRs to viral RNA, leading to inhibition of innate antiviral response. Our findings suggest that hnRNPM acts as an important decoy for excessive innate antiviral immune response.
Assuntos
Proteína DEAD-box 58/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , RNA Viral/metabolismo , Replicação Viral/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Células HEK293 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Ligação Proteica , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/virologia , RNA Viral/genética , Transdução de SinaisRESUMO
Nuclear stress bodies (nSBs) are thermal stress-inducible membrane-less nuclear bodies that are formed on highly repetitive satellite III architectural noncoding RNAs (HSATIII arcRNAs). Upon thermal stress exposure, HSATIII expression is induced to sequestrate specific sets of RNA-binding proteins and form nSBs. The major population of nSBs contain SAFB as a marker, whereas the minor population are SAFB-negative. Here, we found that HNRNPM, which was previously reported to localize in nuclear foci adjacent to SAFB-positive foci upon thermal stress, localizes in a minor population of HSATIII-dependent nSBs. Hence, we used the terms nSB-S and nSB-M to distinguish the SAFB foci and HNRNPM foci, respectively. Analysis of the components of the nSBs revealed that each set contains distinct RNA-binding proteins, including SLTM and NCO5A in nSB-Ss and HNRNPA1 and HNRNPH1 in nSB-Ms. Overall, our findings indicate that two sets of nSBs containing HSATIII arcRNAs and distinct sets of RNA-binding proteins are formed upon thermal stress exposure.
Assuntos
Núcleo Celular/metabolismo , RNA Satélite/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Estresse Fisiológico , Temperatura , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
BACKGROUND: With the popularity of serum prostate-specific antigen (PSA) screening, the number of newly diagnosed prostate cancer (PCa) patients is increasing. However, indolent or invasive PCa cannot be distinguished by PSA levels. Here, we mainly explored the role of heterogeneous nuclear ribonucleoprotein M (hnRNPM) in the invasiveness of PCa. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was used to detect the expressions of hnRNPM in PCa and benign prostate hyperplasia (BPH) tissues as well as in PCa cell lines. Immunohistochemistry was applied to detect the hnRNPM or Yin Yang 1 (YY1) expression in BPH, prostate adenocarcinoma (ADENO) and neuroendocrine prostate cancer (NEPC) tissues. After aberrant, the expression of hnRNPM in C4-2 and PC3 cells, the changes of cell migration and invasion were observed through wound-healing and transwell assays. We also predicted the transcription factor of hnRNPM through databases, then verified the association of hnRNPM and YY1 using chromatin immunoprecipitation (ChIP) and luciferase assays. RESULTS: The expression level of hnRNPM is gradually reduced in BPH, ADENO, and NEPC tissues and it is less expressed in more aggressive PCa cell lines. Overexpression of hnRNPM can significantly reduce Twist1 expression, which inhibits the migration and invasion of PCa cells in vitro. In PCa cells, overexpression of YY1 can promote epithelial-mesenchymal transition by reducing hnRNPM expression. Furthermore, this effect caused by overexpression of YY1 can be partially attenuated by simultaneous overexpression of hnRNPM. CONCLUSIONS: Our study demonstrates that hnRNPM negatively regulated PCa cell migration and invasion, and its expression can be transcriptionally inhibited by YY1. We speculated that hnRNPM may be a biomarker to assist in judging the aggressiveness of PCa.
Assuntos
Adenocarcinoma/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Invasividade Neoplásica/genética , Neoplasias da Próstata/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Masculino , Invasividade Neoplásica/patologia , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
BACKGROUND: Hypoxia suppresses global protein production, yet certain essential proteins are translated through alternative pathways to survive under hypoxic stress. Translation via the internal ribosome entry site (IRES) is a means to produce proteins under stress conditions such as hypoxia; however, the underlying mechanism remains largely uncharacterized. METHODS: Proteomic and bioinformatic analyses were employed to identify hnRNPM as an IRES interacting factor. Clinical specimens and mouse model of tumorigenesis were used for determining the expression and correlation of hnRNPM and its target gene. Transcriptomic and translatomic analyses were performed to profile target genes regulated by hnRNPM. FINDINGS: Hypoxia increases cytosolic hnRNPM binding onto its target mRNAs and promotes translation initiation. Clinical colon cancer specimens and mouse carcinogenesis model showed that hnRNPM is elevated during the development of colorectal cancer, and is associated with poor prognosis. Genome-wide transcriptomics and translatomics analyses revealed a unique set of hnRNPM-targeted genes involved in metabolic processes and cancer neoplasia are selectively translated under hypoxia. INTERPRETATION: These data highlight the critical role of hnRNPM-IRES-mediated translation in transforming hypoxia-induced proteome toward malignancy. FUND: This work was supported by the Ministry of Science and Technology, Taiwan (MOST 104-2320-B-006-042 to HSS and MOST 105-2628-B-001-MY3 to TMC).
Assuntos
Hipóxia Celular , Neoplasias do Colo/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/mortalidade , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos , Fator 9 de Crescimento de Fibroblastos/genética , Fator 9 de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Estimativa de Kaplan-Meier , Camundongos , Biossíntese de Proteínas , Proteínas de Ligação ao Cap de RNA/antagonistas & inibidores , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic analysis integrating three data sources, eCLIP assays for a large RBP panel, shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq, to identify novel such autoregulatory feedback loops. We show that RBPs frequently bind their own pre-mRNAs, their exons respond prominently to NMD pathway disruption, and that the responding exons are enriched with nearby eCLIP peaks. We confirm previously proposed models of autoregulation in SRSF7 and U2AF1 genes and present two novel models, in which (i) SFPQ binds its mRNA and promotes switching to an alternative distal 3'-UTR that is targeted by NMD, and (ii) RPS3 binding activates a poison 5'-splice site in its pre-mRNA that leads to a frame shift and degradation by NMD. We also suggest specific splicing events that could be implicated in autoregulatory feedback loops in RBM39, HNRNPM, and U2AF2 genes. The results are available through a UCSC Genome Browser track hub.
Assuntos
Códon sem Sentido , Degradação do RNAm Mediada por Códon sem Sentido , Splicing de RNA , RNA Interferente Pequeno/metabolismo , Transcriptoma , Regiões 3' não Traduzidas , Processamento Alternativo , Biologia Computacional , Éxons , Mutação da Fase de Leitura , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Spliceossomos , Fator de Processamento U2AF/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer, with a high propensity for distant metastasis and limited treatment options, yet its molecular underpinnings remain largely unknown. Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling protein whose mutations have been causally implicated in several neurologic disorders. Here, we report that a cancer-associated substitution of methionine to isoleucine at residue 276 (M276I) of MORC2 confers gain-of-function properties in the metastatic progression of TNBC. Expression of mutant MORC2 in TNBC cells increased cell migration, invasion, and lung metastasis without affecting cell proliferation and primary tumor growth compared with its wild-type counterpart. The M276I mutation enhanced binding of MORC2 to heterogeneous nuclear ribonucleoprotein M (hnRNPM), a component of the spliceosome machinery. This interaction promoted an hnRNPM-mediated splicing switch of CD44 from the epithelial isoform (CD44v) to the mesenchymal isoform (CD44s), ultimately driving epithelial-mesenchymal transition (EMT). Knockdown of hnRNPM reduced the binding of mutant MORC2 to CD44 pre-mRNA and reversed the mutant MORC2-induced CD44 splicing switch and EMT, consequently impairing the migratory, invasive, and lung metastatic potential of mutant MORC2-expressing cells. Collectively, these findings provide the first functional evidence for the M276I mutation in promoting TNBC progression. They also establish the first mechanistic connection between MORC2 and RNA splicing and highlight the importance of deciphering unique patient-derived mutations for optimizing clinical outcomes of this highly heterogeneous disease.Significance: A gain-of-function effect of a single mutation on MORC2 promotes metastasis of triple-negative breast cancer by regulating CD44 splicing. Cancer Res; 78(20); 5780-92. ©2018 AACR.