RESUMO
The twenty-first century has already recorded more than ten major epidemics or pandemics of viral disease, including the devastating COVID-19. Novel effective antivirals with broad-spectrum coverage are urgently needed. Herein, we reported a novel broad-spectrum antiviral compound PAC5. Oral administration of PAC5 eliminated HBV cccDNA and reduced the large antigen load in distinct mouse models of HBV infection. Strikingly, oral administration of PAC5 in a hamster model of SARS-CoV-2 omicron (BA.1) infection significantly decreases viral loads and attenuates lung inflammation. Mechanistically, PAC5 binds to a pocket near Asp49 in the RNA recognition motif of hnRNPA2B1. PAC5-bound hnRNPA2B1 is extensively activated and translocated to the cytoplasm where it initiates the TBK1-IRF3 pathway, leading to the production of type I IFNs with antiviral activity. Our results indicate that PAC5 is a novel small-molecule agonist of hnRNPA2B1, which may have a role in dealing with emerging infectious diseases now and in the future.
Assuntos
Antivirais , Vírus da Hepatite B , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , SARS-CoV-2 , Animais , Camundongos , Antivirais/farmacologia , COVID-19 , Interferon Tipo I/metabolismo , SARS-CoV-2/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidoresRESUMO
Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycine-rich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3'-untranslated region of HIF-1α mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxia-induced tumor survival and thus offer an avenue for the development of novel anticancer therapies.
Assuntos
Benzofuranos/farmacologia , Produtos Biológicos/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Regiões 3' não Traduzidas , Benzofuranos/química , Produtos Biológicos/química , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Estrutura Molecular , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Estresse Fisiológico/efeitos dos fármacos , Transcrição GênicaRESUMO
Cervical cancer is currently one of the major threats to women's health. The overexpression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as the biomarker has been investigated in various cancers. In our previous study, we found that lobaplatin induced apoptosis and cell cycle arrest via downregulation of proteins including hnRNP A2/B1 in cervical cancer cells. However, the underlying relationship between hnRNP A2/B1 and cervical cancer remained largely unknown. hnRNP A2/B1 knockdown in HeLa and CaSki cells was performed by shRNA transfection. The expression of hnRNP A2/B1 was detected by western blot and Quantitative Realtime PCR. Cell proliferation, migration, invasion and the IC50 of lobaplatin and irinotecan were determined by MTT assay, Transwell assay, Plate colony formation assay and wound healing assay. Flow cytometry was perfomed to investigate cell apoptosis and the cell cycle. The expression of PI3K, AKT, pAKT, p21, p27, caspase3, cleaved caspase3 were revealed by western blot. Nude mouse xenograft model was undertaken with HeLa cells and the xenograft tumor tissue samples were analyzed for the expression of PCNA and Ki67 by immunohistochemistry and the cell morphology was evaluated by hematoxylin and eosin (H&E). Results revealed that hnRNP A2/B1 was successfully silenced in HeLa and CaSki cells. hnRNP A2/B1 knockdown significantly induced the suppression of proliferation, migration, invasion and also enhancement of apoptosis and reduced the IC50 of lobaplatin and irinotecan. The expression of p21, p27 and cleaved caspase3 in shRNA group were significantly upregulated and the expression of pAKT was reduced both in vitro and in vivo. The results of immunohistochemistry showed that PCNA and Ki67 were significantly downregulated in vivo. The growth of nude mouse xenograft tumor was significantly reduced by hnRNP A2/B1 knockdown. Taken together, these data indicate that inhibition of hnRNP A2/B1 in cervical cancer cells can inhibit cell proliferation and invasion, induce cellcycle arrestment and trigger apoptosis via PI3K/AKT signaling pathway. In addition, after silencing hnRNP A2/B1 can increase the sensitivity of cervical cancer cells to lobaplatin and irinotecan.
Assuntos
Biomarcadores Tumorais/metabolismo , Ciclo Celular , Proliferação de Células , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Feminino , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Spinal muscular atrophy (SMA) is caused by deletions or mutations of the Survival Motor Neuron 1 (SMN1) gene coupled with predominant skipping of SMN2 exon 7. The only approved SMA treatment is an antisense oligonucleotide that targets the intronic splicing silencer N1 (ISS-N1), located downstream of the 5' splice site (5'ss) of exon 7. Here, we describe a novel approach to exon 7 splicing modulation through activation of a cryptic 5'ss (Cr1). We discovered the activation of Cr1 in transcripts derived from SMN1 that carries a pathogenic G-to-C mutation at the first position (G1C) of intron 7. We show that Cr1-activating engineered U1 snRNAs (eU1s) have the unique ability to reprogram pre-mRNA splicing and restore exon 7 inclusion in SMN1 carrying a broad spectrum of pathogenic mutations at both the 3'ss and 5'ss of the exon 7. Employing a splicing-coupled translation reporter, we demonstrate that mRNAs generated by an eU1-induced activation of Cr1 produce full-length SMN. Our findings underscore a wider role for U1 snRNP in splicing regulation and reveal a novel approach for the restoration of SMN exon 7 inclusion for a potential therapy of SMA.
Assuntos
Mutação , Sítios de Splice de RNA , Sequências Reguladoras de Ácido Ribonucleico , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Éxons , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Humanos , Íntrons , Camundongos , Atrofia Muscular Espinal/genética , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/biossínteseRESUMO
Increasing evidence has indicated that the splicing factor hnRNPA2B1 plays a direct role in cancer development, progression, gene expression, and signal transduction. Previous studies have shown that knocking down hnRNPA2B1 in breast cancer cells induces apoptosis, but the mechanism and other functions of hnRNPA2B1 in breast cancer are unknown. The goal of this study was to investigate the biological function, clinical significance, and mechanism of hnRNPA2B1 in breast cancer. The expression of hnRNPA2B1 in 92 breast cancer and adjacent normal tissue pairs was analyzed by immunohistochemical staining. Stable clones exhibiting knockdown of hnRNPA2B1 via small hairpin RNA expression were generated using RNA interference technology in breast cancer cell lines. The effects of hnRNPA2B1 on cell proliferation were examined by MTT and EdU assay, and cellular apoptosis and the cell cycle were examined by flow cytometry. A nude mouse xenograft model was established to elucidate the function of hnRNPA2B1 in tumorigenesis in vivo. The role of hnRNPA2B1 in signaling pathways was investigated in vitro. Our data revealed that hnRNPA2B1 was overexpressed in breast cancer tissue specimens and cell lines. Knockdown of hnRNPA2B1 reduced breast cancer cell proliferation, induced apoptosis, and prolonged the S phase of the cell cycle in vitro. In addition, hnRNPA2B1 knockdown suppressed subcutaneous tumorigenicity in vivo. On a molecular level, hnRNPA2B1 knockdown decreased signal transducer and activator of transcription 3 and extracellular-signal-regulated kinase 1/2 phosphorylation. We concluded that hnRNPA2B1 promotes the tumorigenic potential of breast cancer cells, MCF-7 and MDA-MB-231, through the extracellular-signal-regulated kinase 1/2 or signal transducer and activator of transcription 3 pathway, which may serve as a target for future therapies.
Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Fator de Transcrição STAT3/genética , Animais , Apoptose/genética , Neoplasias da Mama/patologia , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/biossíntese , Humanos , Sistema de Sinalização das MAP Quinases/genética , Células MCF-7 , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its end point. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca(2) ](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages, this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Ctsk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Ctsk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Ctsk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss.
Assuntos
Catepsina K/metabolismo , Mitocôndrias/metabolismo , Osteoclastos/metabolismo , Osteogênese , Regiões Promotoras Genéticas , Transdução de Sinais , Animais , Proteína delta de Ligação ao Facilitador CCAAT/antagonistas & inibidores , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Catepsina K/antagonistas & inibidores , Catepsina K/química , Catepsina K/genética , Hipóxia Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genes Reporter , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Camundongos , Mitocôndrias/enzimologia , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/enzimologia , Ligante RANK/metabolismo , Células RAW 264.7 , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ativação TranscricionalRESUMO
Ridaifen-G (RID-G), a tamoxifen analog that we previously synthesized, has potent growth inhibitory activity against various cancer cell lines. Tamoxifen is an anticancer drug known to act on an estrogen receptor (ER) and other proteins. However, our previous studies interestingly suggested that the mechanism of action of RID-G was different from that of tamoxifen. In order to investigate the molecular mode of action of RID-G, we developed a novel chemical genetic approach that combined a phage display screen with a statistical analysis of drug potency and gene expression profiles in thirty-nine cancer cell lines. Application of this method to RID-G revealed that three proteins, calmodulin (CaM), heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), and zinc finger protein 638 (ZNF638) were the candidates of direct targets of RID-G. Moreover, cell lines susceptible to RID-G show similar expression profiles of RID-G target genes. These results suggest that RID-G involves CaM, hnRNP A2/B1, and ZNF638 in its growth inhibitory activity.
Assuntos
Antineoplásicos/química , Tamoxifeno/análogos & derivados , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Técnicas de Patch-Clamp , Biblioteca de Peptídeos , Fosforilação , Ligação Proteica , Proteínas de Ligação a RNA , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Tamoxifeno/química , Tamoxifeno/metabolismo , Tamoxifeno/farmacologia , Fatores de Transcrição , Transcriptoma/efeitos dos fármacosRESUMO
The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Elongação da Transcrição Genética , Processamento Alternativo , Linhagem Celular , Quinase 9 Dependente de Ciclina/metabolismo , Citoplasma/metabolismo , Diclororribofuranosilbenzimidazol/farmacologia , Genes Reporter , Células HCT116 , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Modelos Biológicos , Pressão Osmótica , Fator B de Elongação Transcricional Positiva/metabolismo , Interferência de RNA , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are potent autoantigenic targets in systemic autoimmune rheumatic diseases (SARD). Loss of tolerance to the RA33 complex consisting of hnRNP A2 and its alternatively spliced variants B1 and B2 has been the interest of rheumatologists. A novel ELISA for the detection of anti-hnRNP B1 autoantibodies has been developed to investigate the prevalence thereof in 397 patients with SARD, including patients with rheumatoid arthritis (RA), spondyloarthropathy (SPA), juvenile chronic arthritis, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjögren's syndrome (SS), in comparison to 174 controls. Anti-hnRNP B1 autoantibodies were significantly more prevalent in patients with SARD than controls (47/397, 11.8% versus 2/174, 1.1%; P<0.001). In particular, anti-hnRNP B1 were found more frequently in the disease cohorts than in the controls and were present in 24/165 (14.5%) patients with RA, 6/58 (10.3%) SPA, 11/65 (16.9%) SSc, and 4/50 (8.0%) SLE. In RA patients, anti-hnRNP B1 autoantibodies correlated significantly with C-reactive protein levels and erythrocyte sedimentation rate, while in patients with SSc it was associated with features of arterial wall stiffness and presence of hypertension. Anti-hnRNP B1 autoantibodies occur in SARD and seem to be correlated with distinct clinical characteristics in patients with RA and SSc.
Assuntos
Artrite Reumatoide/fisiopatologia , Autoanticorpos/sangue , Autoantígenos/sangue , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/sangue , Escleroderma Sistêmico/fisiopatologia , Adolescente , Adulto , Artrite Juvenil/diagnóstico , Artrite Juvenil/imunologia , Artrite Juvenil/fisiopatologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Sedimentação Sanguínea , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Humanos , Hipertensão/fisiopatologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Pessoa de Meia-Idade , Federação Russa , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/imunologia , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/fisiopatologia , Espondiloartropatias/diagnóstico , Espondiloartropatias/imunologia , Espondiloartropatias/fisiopatologia , Rigidez VascularRESUMO
Quercetin, a flavonoid abundantly present in plants, is widely used as a phytotherapy in prostatitis and prostate cancer. Although quercetin has been reported to have a number of therapeutic effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and in vitro binding experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules after treatment of cells with quercetin for up to 48 h, and the levels of cIAP1 (cellular inhibitor of apoptosis), an internal ribosome entry site translation-dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1, insights that were obtained using a chemical proteomics strategy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Quercetina/farmacologia , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/farmacocinética , Apoptose/efeitos dos fármacos , Sítios de Ligação , Transporte Biológico Ativo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fitoterapia , Neoplasias da Próstata/genética , Ligação Proteica , Proteômica , Quercetina/farmacocinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta Carioferinas/metabolismoRESUMO
In recent years, it has become clear that splicing factors play a direct role in cancer development. We showed previously that splicing factors SRSF1, SRSF6, and hnRNP A2/B1 are up-regulated in several cancers and can act as oncogenes when up-regulated. Here we examined the role of splicing factors hnRNP A1/A1b and hnRNP A2/B1 in hepatocellular carcinoma (HCC). We show that the splicing factors hnRNP A1 and hnRNP A2 are up-regulated in HCC tumors derived from inflammation-induced liver cancer mouse model. Overexpression of hnRNP A1 or hnRNP A2, but not the splicing isoform hnRNP B1, induced tumor formation of immortalized liver progenitor cells, while knockdown of these proteins inhibited anchorage-independent growth and tumor growth of human liver cancer cell lines. In addition, we found that cells overexpressing hnRNP A2 showed constitutive activation of the Ras-MAPK-ERK pathway. In contrast, knockdown of hnRNP A2 inhibited the Ras-MAPK-ERK pathway and prevented ERK1/2 activation by EGF. Moreover, we found that hnRNP A2 regulates the splicing of A-Raf, reducing the production of a short dominant-negative isoform of A-Raf and elevating the full-length A-Raf transcript. Taken together, our data suggest that hnRNP A2 up-regulation in HCC induces an alternative splicing switch that down-regulates a dominant-negative isoform of A-Raf, leading to activation of the Raf-MEK-ERK pathway and cellular transformation.
Assuntos
Processamento Alternativo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas A-raf/genética , Proteínas ras/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Animais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Hepatócitos/metabolismo , Hepatócitos/patologia , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Inflamação/complicações , Inflamação/genética , Inflamação/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Natural products are the major sources of currently available anticancer drugs. We recently reported that phenanthrene-based tylophorine derivative-1 (PBT-1) may be a potential antitumor agent for lung adenocarcinoma. We therefore examined the direct targets of PBT-1 and their effects in inhibiting lung adenocarcinoma. We found that PBT-1 reduced the level of Slug and inhibits the migration, invasion, and filopodia formation of lung adenocarcinoma CL1-5 cells in vitro. In addition, PBT-1 displayed in vivo antitumor and antimetastasis activities against subcutaneous and orthotopic xenografts of CL1-5 cells in nude mice. Chemical proteomics showed that heat shock protein 90 (HSP90) and heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) bound PBT-1 in CL1-5 cells. Inhibition of HSP90 and hnRNP A2/B1 reduced the activation of AKT and Slug expression. Taken together, these findings suggest that PBT-1 binds to HSP90 and/or hnRNP A2/B1 and initiates antitumor activities by affecting Slug- and AKT-mediated metastasis and tumorigenesis.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Ácido 8,11,14-Eicosatrienoico/farmacologia , Ácido 8,11,14-Eicosatrienoico/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/uso terapêutico , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/patologiaRESUMO
Heterogeneous nuclear ribonucleoparticule A1/A2 (hnRNP A1/A2) and splicing factor 2/alternative splicing factor (SF2/ASF) are pivotal for precursor messenger RNA (pre-mRNA) splicing. Interferon regulatory factor-3 (IRF-3) plays critical roles in host defense against viral and microbial infection. Truncated IRF-3 proteins resulting from alternative splicing have been identified and characterized as functional antagonists to full-length IRF-3. In this study, we examined the molecular mechanism for splicing regulation of IRF-3 pre-mRNA and first reported the regulatory effect of hnRNP A1/A2 and SF2/ASF on IRF-3 splicing and activation. RNA interference-mediated depletion of hnRNP A1/A2 or SF2/ASF in human non-small cell lung cancer (NSCLC) cells increased exclusion of exons 2 and 3 of IRF-3 gene and reduced expression levels of IRF-3 protein and IRF-3 downstream effector molecules interferon-beta and CXCL10/IP-10. In addition, direct binding of hnRNP A1 and SF2/ASF to specific binding motifs in IRF-3 intron 1 was confirmed by RNA electrophoretic mobility shift assay. Subsequent minigene splicing assay showed that IRF-3 minigenes with mutated hnRNPA 1/A2 or SF2/ASF binding motifs increased exclusion of exons 2 and 3. Moreover, knockdown of hnRNP A1/A2 or SF2/ASF in NSCLC cells reinforced phytohemagglutinin-induced tumor necrosis factor-alpha release by peripheral blood mononuclear cells (PBMC) but suppressed that of interleukin-10 in NSCLC/PBMC co-cultures. Taken together, our results suggest that specific knockdown for hnRNP A1/A2 or SF2/ASF increase exclusion of exons 2 and 3 of IRF-3 pre-mRNA and influence immunomodulatory functions of human NSCLC cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/imunologia , Fator Regulador 3 de Interferon/imunologia , Neoplasias Pulmonares/imunologia , Proteínas Nucleares/imunologia , Precursores de RNA/imunologia , Proteínas de Ligação a RNA/imunologia , Processamento Alternativo , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Técnicas de Cocultura , Éxons , Regulação da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Imunomodulação , Fator Regulador 3 de Interferon/genética , Interferon beta/genética , Interferon beta/imunologia , Íntrons , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Dados de Sequência Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Precursores de RNA/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina , Transdução de SinaisRESUMO
We investigated the effects of inhibiting heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) expression on apoptosis, invasion, migration, and the chemotherapy sensitivity of pancreatic cancer cells to gemcitabine, 5-FU, and oxaliplatin chemotherapy using small interfering RNA (siRNA). Chemically synthesized siRNA hnRNP A2/B1 was transfected into the human pancreatic cancer cell lines SW1990 and BxPC-3. The IC(50) of gemcitabine, 5-FU, and oxaliplatin was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and cycle were detected using flow cytometry. The expressions of apoptosis-related genes, p53, Bax, Bcl-2, TRAIL, Survivin, multidrug resistance 1 (MDR1), E-cadherin, and matrix metalloproteinases-2 (MMP-2) were detected using real-time PCR and western blot. Plate colony formation assay, wound scratch assay, invasion, and migration were also examined. Gemcitabine, 5-FU, and oxaliplatin inhibit the proliferation of SW1990 and BxPC-3 cells in a concentration-dependent manner. Inhibition of hnRNP A2/B1 expression significantly reduced the IC(50) of gemcitabine, 5-FU, and oxaliplatin (P<0.01). hnRNP A2/B1 siRNA combined with gemcitabine, 5-FU and oxaliplatin significantly increased (P<0.01) apoptosis of pancreatic cancer cell lines SW1990 and BxPC-3, increased the expression level of Bax mRNA, decreased Bcl-2 mRNA and MDR1 mRNA expression (P<0.01), and induced no change in p53, TRAIL, and Survivin mRNA expression in SW1990. In the western blot analysis, the expression level of Bax protein increased (P<0.01); the expression of both P-glycoprotein (Pg-p) protein and Bcl-2 protein decreased (P<0.01). Silencing hnRNP A2/B1 decreased invasion and migration in the cell line SW1990. Silencing hnRNP A2/B1 in SW1990 also correlated with an increase in E-cadherin expression and a decrease in MMP-2 expression at the same time. Inhibition of hnRNP A2/B1 expression can induce apoptosis in pancreatic cancer cells and improve chemosensitivity to gemcitabine, 5-FU, and oxaliplatin. hnRNP A2/B1 may play a role in invasion and migration in the pancreatic cancer cell line SW1990 through the regulation of E-cadherin and expression of MMP-2.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Fluoruracila/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Neoplásicos/efeitos dos fármacos , Humanos , Compostos Organoplatínicos/farmacologia , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Fas is a transmembrane cell surface protein recognized by Fas ligand (FasL). When FasL binds to Fas, the target cells undergo apoptosis. A soluble Fas molecule that lacks the transmembrane domain is produced from skipping of exon 6 encoding this region in alternative splicing procedure. The soluble Fas molecule has the opposite function of intact Fas molecule, protecting cells from apoptosis. Here we show that knockdown of hnRNP A1 promotes exon 6 skipping of Fas pre-mRNA, whereas overexpression of hnRNP A1 reduces exon 6 skipping. Based on the bioinformatics approach, we have hypothesized that hnRNP A1 functions through interrupting 5' splice site selection of exon 5 by interacting with its potential binding site close to 5' splice site of exon 5. Consistent with our hypothesis, we demonstrate that mutations of the hnRNP A1 binding site on exon 5 disrupted the effects of hnRNP A1 on exon 6 inclusion. RNA pull-down assay and then western blot analysis with hnRNP A1 antibody prove that hnRNP A1 contacts the potential binding site RNA sequence on exon 5 but not the mutant sequence. In addition, we show that the mutation of 5' splice site on exon 5 to a less conserved sequence destructed the effects of hnRNP A1 on exon 6 inclusion. Therefore we conclude that hnRNP A1 interacts with exon 5 to promote distal exon 6 inclusion of Fas pre-mRNA. Our study reveals a novel alternative splicing mechanism of Fas pre-mRNA.
Assuntos
Apoptose/genética , Éxons , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Precursores de RNA/genética , Receptor fas/genética , Processamento Alternativo , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Íntrons , Dados de Sequência Molecular , Ligação Proteica , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Homologia de Sequência do Ácido Nucleico , Receptor fas/metabolismoRESUMO
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was reported to be participated in tumor development. The association between hnRNP A1 and liver cancer and the functional role of hnRNP A1 in liver cancer have never been reported. Herein, hnRNP A1-specific single-stranded DNA aptamer, BC15, was used to (a) evaluate hnRNP A1 expression in liver cancer, and (b) treat hepatocarcinoma by acting as an inhibitor of hnRNP A1. Results showed that there is high hnRNP A1 expression in liver cancer including serum α-fetoprotein-negative liver cancer tissues compared with either para-cancer or benign controls. Down regulation of hnRNP A1 expression by RNA interference inhibits the proliferation and migration of cancerous HepG2 cells, while overexpression of hnRNP A1 in normal HL-7702 cells increased the proliferation and migration of the cells. Importantly, BC15 showed a stronger inhibiting effect on the proliferation of cultured hepatoma cells than hnRNP A1 small interfering RNA, strongly suggesting that BC15 could also be a potential drug candidate for an hnRNP A1 inhibitor besides its prospect utility in in situ histological examination.
Assuntos
Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Células Hep G2 , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42.
Assuntos
Expressão Gênica , HIV-1/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/fisiologia , HIV-1/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/fisiologia , Humanos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Interferência de RNA , RNA Viral/análise , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 µM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Inibidores da Topoisomerase I/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Drosophila melanogaster , Técnicas de Inativação de Genes , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Técnicas de Microbalança de Cristal de QuartzoRESUMO
PURPOSE: The Src family kinase Fyn, heterogenous nuclear ribonucleoprotein (HnRNP) A2B1 and Sam68 are thought to be associated with the metastasis of tumors, but their roles in the regulation of apoptosis remain unclear. This study investigated the role of Fyn and its potential relationship with HnRNPA2B1 and Sam68 in the regulation of apoptosis in pancreatic cancer. Experimental design. We examined both the activity of Fyn and the expression of HnRNPA2B1 in human pancreatic cancer tissues and systematically investigated the apoptotic mechanisms induced by Fyn activity using multiple experimental approaches. RESULTS: We found that Fyn activity was increased in metastatic pancreatic cancer tissues. In the pancreatic cancer BxPc3 cell line, the inhibition of Fyn activity by kinase-dead Fyn downregulated HnRNPA2B1 expression. Further analysis showed that HnRNPA2B1 expression was associated with pancreatic cancer progression. In BxPc3 cells, HnRNPA2B1 bound to Bcl-x messenger RNA (mRNA), which affected splicing and therefore, the formation of Bcl-x(s). Downregulation of HnRNPA2B1 by RNA interference (RNAi) resulted in the increased formation of the pro-apoptotic Bcl-x(s) and promoted apoptosis of BxPc3 cells. In addition, deactivation of Fyn in BxPc3 cells reduced Sam68 phosphorylation. This resulted in increased binding between Sam68 and Bcl-x mRNA, promoting the formation of the anti-apoptotic Bcl-x(L). The knockdown of Sam68 by RNAi also increased the formation of Bcl-x(L). Finally, HnRNPA2B1 overexpression or Sam68 knockdown could rescue pancreatic cancer cells from apoptosis. CONCLUSION: Our results suggest a mechanism by which Fyn requires HnRNPA2B1 and Sam68 to coordinate and regulate apoptosis, thus promoting the proliferation and metastasis of pancreatic cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Neoplasias Pancreáticas/genética , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Nuclear factor TDP-43 has been reported to play multiple roles in transcription, pre-mRNA splicing, mRNA stability and mRNA transport. From a structural point of view, TDP-43 is a member of the hnRNP protein family whose structure includes two RRM domains flanked by the N-terminus and C-terminal regions. Like many members of this family, the C-terminal region can interact with cellular factors and thus serve to modulate its function. Previously, we have described that TDP-43 binds to several members of the hnRNP A/B family through this region. In this work, we set up a coupled minigene/siRNA cellular system that allows us to obtain in vivo data to address the functional significance of TDP-43-recruited hnRNP complex formation. Using this method, we have finely mapped the interaction between TDP-43 and the hnRNP A2 protein to the region comprised between amino acid residues 321 and 366. Our results provide novel details of protein-protein interactions in splicing regulation. In addition, we provide further insight on TDP-43 functional properties, particularly the lack of effects, as seen with our assays, of the disease-associated mutations that fall within the TDP-43 321-366 region: Q331K, M337V and G348C.