RESUMO
Glioblastoma (GBM) represents a primary malignant brain tumor. Temozolomide resistance is a major hurdle in GBM treatment. Proteins encoded by circular RNAs (circRNAs) can modulate the sensitivity of multiple tumor chemotherapies. However, the impact of circRNA-encoded proteins on GBM sensitivity to temozolomide remains unknown. Herein, we discover a circRNA (circCOPA) through the circRNA microarray profile in GBM samples, which can encode a novel 99 amino acid protein (COPA-99aa) through its internal ribosome entry site. Functionally, circCOPA overexpression in GBM cells inhibits cell proliferation, migration, and invasion in vitro and growth in vivo. Rather than itself, circCOPA mainly functions as a suppressive effector by encoding COPA-99aa. Moreover, we reveal that circCOPA is downregulated in GBM tissues and high expression of circCOPA is related to a better prognosis in GBM patients. Mechanistically, a heteromer of SFPQ and NONO is required for double-strand DNA break repair. COPA-99aa disrupts the dimerization of NONO and SFPQ by separately binding with the NONO and SFPQ proteins, thus resulting in the inhibition of proliferation or invasion and the increase of temozolomide-induced DNA damage in GBM cells. Collectively, our data suggest that circCOPA mainly contributes to inhibiting the GBM malignant phenotype through its encoded COPA-99aa and that COPA-99aa increases temozolomide-induced DNA damage by interfering with the dimerization of NONO and SFPQ. Restoring circCOPA or COPA-99aa may increase the sensitivity of patients to temozolomide.
Assuntos
Neoplasias Encefálicas , Proliferação de Células , Glioblastoma , RNA Circular , Proteínas de Ligação a RNA , Temozolomida , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , RNA Circular/genética , RNA Circular/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Animais , Fator de Processamento Associado a PTB/metabolismo , Fator de Processamento Associado a PTB/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fenótipo , Movimento Celular/efeitos dos fármacos , Camundongos , Reparo do DNA/efeitos dos fármacos , Camundongos Nus , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos Alquilantes/farmacologiaRESUMO
In this study, we aimed to investigate cytoplasmic maturation and miRNA expression of mature oocytes cultured in porcine follicular fluid exosomes. We also examined the effect of miR-339-5p on oocyte maturation. Twenty eight differentially expressed miRNAs were detected using miRNA-seq. We then transfected cumulus oocyte complexes with miR-339-5p mimics and inhibitor during culture. The results showed that exosomes increased endoplasmic reticulum levels and the amount of lipid droplets, and decreased ROS levels, lipid droplet size, and percentage of oocytes with abnormal cortical granule distribution. Overexpressing miR-339-5p significantly decreased cumulus expansion genes, oocyte maturation-related genes, target gene proline/glutamine-rich splicing factor (SFPQ), ERK1/2 phosphorylation levels, oocyte maturation rate, blastocyst rate, and lipid droplet number, but increased lipid droplet size and the ratio of oocytes with abnormal cortical granule distribution. Inhibiting miR-339-5p reversed the decrease observed during overexpression. Mitochondrial membrane potential and ROS levels did not differ significantly between groups. In summary, exosomes promote oocyte cytoplasmic maturation and miR-339-5p regulating ERK1/2 activity through SFPQ expression, thereby elevating oocyte maturation and blastocyst formation rate in vitro.
Assuntos
Exossomos , Líquido Folicular , Técnicas de Maturação in Vitro de Oócitos , Sistema de Sinalização das MAP Quinases , MicroRNAs , Oócitos , Animais , Suínos , MicroRNAs/metabolismo , MicroRNAs/genética , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Exossomos/metabolismo , Feminino , Líquido Folicular/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Fator de Processamento Associado a PTB/genética , Regulação da Expressão GênicaRESUMO
The MUC1 gene evolved in mammals for adaptation of barrier tissues in response to infections and damage. Paraspeckles are nuclear bodies formed on the NEAT1 lncRNA in response to loss of homeostasis. There is no known intersection of MUC1 with NEAT1 or paraspeckles. Here, we demonstrate that the MUC1-C subunit plays an essential role in regulating NEAT1 expression. MUC1-C activates the NEAT1 gene with induction of the NEAT1_1 and NEAT1_2 isoforms by NF-κB- and MYC-mediated mechanisms. MUC1-C/MYC signaling also induces expression of the SFPQ, NONO and FUS RNA binding proteins (RBPs) that associate with NEAT1_2 and are necessary for paraspeckle formation. MUC1-C integrates activation of NEAT1 and RBP-encoding genes by recruiting the PBAF chromatin remodeling complex and increasing chromatin accessibility of their respective regulatory regions. We further demonstrate that MUC1-C and NEAT1 form an auto-inductive pathway that drives common sets of genes conferring responses to inflammation and loss of homeostasis. Of functional significance, we find that the MUC1-C/NEAT1 pathway is of importance for the cancer stem cell (CSC) state and anti-cancer drug resistance. These findings identify a previously unrecognized role for MUC1-C in the regulation of NEAT1, RBPs, and paraspeckles that has been co-opted in promoting cancer progression.
Assuntos
Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Mucina-1 , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Mucina-1/genética , Mucina-1/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/metabolismo , Transdução de Sinais/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a DNARESUMO
Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells, as well as of Kaposi's sarcoma-associated herpesvirus in B cells, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency.
Assuntos
Herpesvirus Humano 4 , Histonas , Fator de Processamento Associado a PTB , Ativação Viral , Latência Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Histonas/metabolismo , Ativação Viral/genética , Latência Viral/genética , Fator de Processamento Associado a PTB/metabolismo , Fator de Processamento Associado a PTB/genética , Regulação Viral da Expressão Gênica , Linfócitos B/virologia , Linfócitos B/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Sistemas CRISPR-Cas , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Transativadores/genética , Genoma ViralRESUMO
Neurodegenerative diseases are commonly classified as proteinopathies that are defined by the aggregation of a specific protein. Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are classified as synucleinopathies since α-synuclein (α-syn)-containing inclusions histopathologically define these diseases. Unbiased biochemical analysis of PD and DLB patient material unexpectedly revealed novel pathological inclusions in the nucleus comprising adenosine-to-inosine (A-to-I)-edited mRNAs and NONO and SFPQ proteins. These inclusions showed no colocalization with Lewy bodies and accumulated at levels comparable to α-syn. NONO and SFPQ aggregates reduced the expression of the editing inhibitor ADAR3, increasing A-to-I editing mainly within human-specific, Alu-repeat regions of axon, synaptic, and mitochondrial transcripts. Inosine-containing transcripts aberrantly accumulated in the nucleus, bound tighter to recombinant purified SFPQ in vitro, and potentiated SFPQ aggregation in human dopamine neurons, resulting in a self-propagating pathological state. Our data offer new insight into the inclusion composition and pathophysiology of PD and DLB.
Assuntos
Doença por Corpos de Lewy , Fator de Processamento Associado a PTB , Doença de Parkinson , Edição de RNA , Humanos , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Doença por Corpos de Lewy/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fator de Processamento Associado a PTB/metabolismo , Fator de Processamento Associado a PTB/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Inosina/metabolismo , Adenosina/metabolismo , Núcleo Celular/metabolismo , Masculino , Idoso , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Feminino , RNA Mensageiro/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Idoso de 80 Anos ou maisRESUMO
The cellular SFPQ protein is involved in several stages of the HIV-1 life cycle, but the detailed mechanism of its involvement is not yet fully understood. Here, the role of SFPQ in the early stages of HIV-1 replication has been studied. It is found that changes in the intracellular level of SFPQ affect the integration of viral DNA, but not reverse transcription, and SFPQ is a positive factor of integration. A study of the SFPQ interaction with HIV-1 integrase (IN) has revealed two diRGGX1-4 motifs in the N-terminal region of SFPQ, which are involved in IN binding. Substitution of a single amino acid residue in any of these regions led to a decrease in binding efficiency, while mutations in both motifs almost completely disrupted the SFPQ interaction with IN. The effect of the SFPQ mutants with impaired ability to bind IN on viral replication has been analyzed. Unlike the wild-type protein, the SFPQ mutants did not affect viral integration. This confirms that SFPQ influences the integration stage through direct interaction with IN. Our results indicate that the SFPQ/IN complex can be considered as a potential therapeutic target for the development of new inhibitors of HIV replication.
Assuntos
Integrase de HIV , HIV-1 , Fator de Processamento Associado a PTB , Integração Viral , Replicação Viral , HIV-1/metabolismo , HIV-1/fisiologia , HIV-1/genética , Humanos , Integrase de HIV/metabolismo , Integrase de HIV/genética , Fator de Processamento Associado a PTB/metabolismo , Fator de Processamento Associado a PTB/genética , Ligação Proteica , Mutação , Células HEK293RESUMO
Splicing factor proline- and glutamine-rich (SFPQ) can interact with RNAs to regulate gene expression. The function of SFPQ in the immunotherapy of non-small cell lung cancer (NSCLC) is investigated in this study. H1299 and A549 cells were transfected with shSFPQ plasmid. Cell counting kit-8 (CCK-8) and cell clone formation were utilized to detect survival and proliferation. Programmed death-ligand 1 (PD-L1) and SFPQ were detected in NSCLC patients treated with anti-PD-L1 antibody. Dual-luciferase assays, RNA immunoblotting, RNA pull-down, and mRNA stability assay were applied to verify the regulation of PD-L1 with SFPQ. Human peripheral blood mononuclear cells (PBMC)-derived dendritic cells were loaded with irradiated A549 and H1299 cells, which were cultured with autologous CD8+T cells and tumor cells to perform in vitro tumor-specific cytotoxic T lymphocytes (CTL) cytotoxicity analysis. SFPQ silencing inhibited the survival and proliferation of H1299 and A549 cells with down-regulated PD-L1 expression. PD-L1 and SFPQ expression were markedly higher in anti-PD-L1 antibody treatment responders compared to non-responders, which showed a positive Pearson correlation (Râ =â 0.76, Pâ <â .001). SFPQ up-regulated the relative mRNA and protein expression of PD-L1 by binding to the PD-L1 3'UTR to slow the decay of PD-L1 mRNA. SFPQ silencing promoted the killing effect of CTL on A549 and H1299 cells. SFPQ up-regulates PD-L1 expression by binding with PD-L1 3'UTR to slow the decay of PD-L1 mRNA, and SFPQ silencing promotes CTL-mediated cytotoxicity on NSCLC cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Regiões 3' não Traduzidas , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Carcinoma Pulmonar de Células não Pequenas/patologia , Glutamina , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo , Fatores de Processamento de RNA/genética , Linfócitos T Citotóxicos/metabolismo , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/metabolismoRESUMO
Human polypyrimidine-binding splicing factor (PSF/SFPQ) is a tumor suppressor protein that regulates the gene expression of several proto-oncogenes and binds to the 5'-polyuridine negative-sense template (5'-PUN) of some RNA viruses. The activity of PSF is negatively regulated by long-noncoding RNAs, human metastasis associated in lung adenocarcinoma transcript-1 and murine virus-like 30S transcript-1 (VL30-1). PSF is a 707-amino acid protein that has a DNA-binding domain and two RNA recognition motifs (RRMs). Although the structure of the apo-truncated PSF is known, how PSF recognizes RNA remains elusive. Here, we report the 2.8 Å and 3.5 Å resolution crystal structures of a biologically active truncated construct of PSF (sPSF, consisting of residues 214-598) alone and in a complex with a 30mer fragment of VL30-1 RNA, respectively. The structure of the complex reveals how the 30mer RNA is recognized at two U-specific induced-fit binding pockets, located at the previously unrecognized domain-swapped, inter-subunit RRM1 (of the first subunit)-RRM2 (of the second subunit) interfaces that do not exist in the apo structure. Thus, the sPSF dimer appears to have two conformations in solution: one in a low-affinity state for RNA binding, as seen in the apo-structure, and the other in a high-affinity state for RNA binding, as seen in the sPSF-RNA complex. PSF undergoes an all or nothing transition between having two or no RNA-binding pockets. We predict that the RNA binds with a high degree of positive cooperativity. These structures provide an insight into a new regulatory mechanism that is likely involved in promoting malignancies and other human diseases.
Assuntos
RNA Longo não Codificante , Proteínas de Ligação a RNA , Animais , Humanos , Camundongos , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/metabolismo , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismoAssuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Fator de Processamento Associado a PTB/efeitos dos fármacos , RNA Longo não Codificante/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Tratamento Farmacológico/métodos , Tratamento Farmacológico/estatística & dados numéricos , Humanos , Fator de Processamento Associado a PTB/genética , RNA Longo não Codificante/farmacologiaRESUMO
RNA-binding protein PSF functions as an epigenetic modifier by interacting with long noncoding RNAs and the corepressor complex. PSF also promotes RNA splicing events to enhance oncogenic signals. In this study, we conducted an in vitro chemical array screen and identified multiple small molecules that interact with PSF. Several molecules inhibited RNA binding by PSF and decreased prostate cancer cell viability. Among these molecules and its derivatives was a promising molecule, No. 10-3 [7,8-dihydroxy-4-(4-methoxyphenyl)chromen-2-one], that was the most effective at blocking PSF RNA-binding ability and suppressing treatment-resistant prostate and breast cancer cell proliferation. Exposure to No. 10-3 inhibited PSF target gene expression at the mRNA level. Treatment with No. 10-3 reversed epigenetically repressed PSF downstream targets, such as cell-cycle inhibitors, at the transcriptional level. Chromatin immunoprecipitation sequencing in prostate cancer cells revealed that No. 10-3 enhances histone acetylation to induce expression of apoptosis as well as cell-cycle inhibitors. Furthermore, No. 10-3 exhibited antitumor efficacy in a hormone therapy-resistant prostate cancer xenograft mouse model, suppressing treatment-resistant tumor growth. Taken together, this study highlights the feasibility of targeting PSF-mediated epigenetic and RNA-splicing activities for the treatment of aggressive cancers. SIGNIFICANCE: This study identifies small molecules that target PSF-RNA interactions and suppress hormone therapy-refractory cancer growth, suggesting the potential of targeting PSF-mediated gene regulation for cancer treatment.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Epigênese Genética , Fator de Processamento Associado a PTB/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fator de Processamento Associado a PTB/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Longo não Codificante/genética , Transcrição Gênica , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Reactive oxygen species (ROS), a by-product of oxygen metabolism mainly originating from mitochondria, participate in many pathological processes related to ophthalmopathy. Excessive production of ROS leads to oxidative stress, which influences the permeability, proliferation, migration, and tube formation of human retinal microcapillary endothelial cells (HRMECs). The molecular mechanisms underlying the effects of ROS are not clear. In Vldlr-/- mice, we used fundus fluorescein angiography and retinal flat mount staining to observe the effect of polypyrimidine tract-binding protein-associated splicing factor (PSF) on pathological retinal neovascularization in vivo. Additionally, in human retinal microvascular endothelial cells treated with 4-HNE, cell viability, tube formation, wound healing, and Transwell assays were performed to study the effect of PSF on the proliferation, migration, and tube formation of retinal vascular endothelial cells in vitro. Moreover, reactive oxygen species assay, real-time PCR, and Western blot were included to analyze the potential mechanism of PSF in the above series of effects. PSF ameliorated intraretinal neovascularization (IRNV) in vivo in Vldlr-/- mice. Under 4-hydroxynonenal (4-HNE) conditions in vitro, PSF reprogrammed mitochondrial bioenergetic and glycolytic profiles. It also reduced ROS levels and inhibited 4-HNE-induced angiogenesis, which involves the proliferation, migration, and tube formation of HRMECs. These results suggest that PSF participates in the regulation of HRMECs proliferation and migration during the development of pathological angiogenesis. We demonstrated that PSF enhanced Nrf2 activation and heme oxygenase-1 (HO-1) expression via extracellular signal-regulated kinase (ERK) and Akt signaling in HRMECs, which subsequently resulted in intracellular ROS scavenging. PSF restored endoplasmic reticulum (ER) redox homeostasis, which was indicated by an increase in protein disulfide isomerase (PDI) and Ero-1α and a reduction in GRP78 and C/EBP homologous protein (CHOP). PSF also attenuated ER stress via regulation of the protein kinase R (PKR)-like endoplasmic reticulum kinase PERK/eukaryotic translation factor 2 alpha (eIF2α)/activating transcription factor 4 (ATF4) pathway in 4-HNE-treated HRMECs. Our research shows that PSF may be a potential antioxidant that regulates pathological angiogenesis through ERK-AKT/Nrf2/HO-1 and PERK/eIF2α/ATF4 signal regulation. KEY MESSAGES: Reactive oxygen species (ROS) mainly originating from mitochondria is a by-product of oxygen metabolism in the body and participates in the pathological process related to multiple blindness-related ophthalmopathy. Moreover , excessive production of ROS will lead to oxidative stress. Consequently, oxidative stress influences the permeability, proliferation, migration, and tube formation of human retinal microcapillary endothelial cells (HRMECs). The molecular mechanisms underlying the effects of ROS remain unclear. Here, we reveal that Polypyrimidine tract-binding protein-associated splicing factor (PSF) ameliorates intraretinal neovascularization (IRNV) in vivo in Vldlr-/- mice. Furthermore, under 4-HNE conditions in vitro, PSF reprograms mitochondrial bioenergetic and glycolytic profiles, reduces ROS levels, and inhibits 4-HNE-induced angiogenesis, which involves the proliferation, migration, and tube formation of HRMECs, suggesting that it participates in regulating the proliferation and migration of HRMECs during the development of pathological angiogenesis. Furthermore, PSF enhances Nrf2 activation and HO-1 expression through ERK and AKT signaling in HRMECs, resulting in intracellular ROS scavenging. PSF restores endoplasmic reticulum (ER) redox homeostasis, as indicated by an increase in PDI and Ero-1α and a reduction in GRP78 and CHOP. PSF also attenuates ER stress by regulating the PERK/eIF2α/ATF4 pathway in 4-HNE-treated HRMECs.
Assuntos
Fator de Processamento Associado a PTB/metabolismo , Neovascularização Retiniana/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Aldeídos/farmacologia , Animais , Células Cultivadas , Estresse do Retículo Endoplasmático , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/citologia , Mitocôndrias/metabolismo , Fator de Processamento Associado a PTB/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/genética , Retina/citologia , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/genética , eIF-2 Quinase/metabolismoRESUMO
The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These SFPQ-inhibited CLEs appear preferentially in long introns of genes with neuronal functions and can dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found expressed across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a gene regulation mechanism with wide relevance to human neuropathologies.
Assuntos
Esclerose Lateral Amiotrófica/genética , Códon sem Sentido , Éxons/genética , Fator de Processamento Associado a PTB/genética , Animais , Sequência de Bases , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Hibridização In Situ/métodos , Íntrons/genética , Camundongos , Neurônios/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.
Assuntos
Esclerose Lateral Amiotrófica/genética , Ciclinas/genética , Demência Frontotemporal/genética , Fator de Processamento Associado a PTB/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Células HEK293 , Humanos , Agregados Proteicos/genética , Mapas de Interação de Proteínas/genética , Proteólise , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genéticaRESUMO
BACKGROUND: Abnormal neovascularization is the most common cause of blindness, and hypoxia alters tissue metabolism, function, and morphology. HIF-1α, the transcriptional activator of VEGF, has intricate mechanisms of nuclear translocation and activation, but its signal termination mechanisms remain unclear. METHODS: We investigated the role of polypyrimidine tract-binding protein-associated splicing factor (PSF) in cellular energy production, migration, and proliferation by targeting HIF-1α in vivo and in vitro PSF plasmids were transfected with liposome 2000 transfection reagent. Young C57/BL6J mice were kept in a hyperoxia environment, followed by indoor air, resulting in oxygen-induced retinopathy. Oxygen-induced retinopathy (OIR) animals were randomly divided into three groups: OIR group, OIR + vector group (OIR cubs treated with rAAV vector) and OIR + PSF group (OIR cubs treated with rAAV-PSF). Age-matched C57/BL6J mice were used as controls and exposed to constant normoxic conditions. The animals were executed and their pupils were subjected to subsequent experiments. The metabolic spectrum was analyzed by Seahorse XFe96 flux analyzer, and OCR and extracellular acidification rate were quantified at the same time. RESULTS: PSF ameliorated retinal neovascularization and corrected abnormal VEGF expression in mice with oxygen-induced retinopathy and reduced intra-retinal neovascularization in Vldlr - / - mice. PSF reprogrammed mitochondrial bioenergetics and inhibited the transition of endothelial cells after hypoxia, suggesting its involvement in pathological angiogenesis.Ectopic PSF expression inhibited hypoxia-induced HIF-1α activation in the nucleus by recruiting Hakai to the PSF/HIF-1α complex, causing HIF-1α inhibition. PSF knockdown increased hypoxia-stimulated HIF-1α reactions. These hypoxia-dependent processes may play a vital role in cell metabolism, migration, and proliferation. Thus, PSF is a potential treatment target in neovascularization-associated ophthalmopathy. CONCLUSION: This is the first study showing that PSF inhibits HIF-1α via recruitment of Hakai, modulates mitochondrial oxidation and glycolysis, and downregulates VEGF expression under hypoxia. We propose a new HIF-1 α/Hakai regulatory mechanism that may play a vital role in the pathogenesis of neovascularization in ophthalmopathy. PSF-Hakai-HIF-1α signaling pathway under hypoxia condition. Schematic diagram showing that the PSF-Hakai-HIF-1α signaling pathway. Under hypoxia condition, PSF-Hakai complex regulate HIF-1α signaling, thus inhibiting downstream target gene VEGF, cell metabolism and angiogenesis eventually. Video Abstract: Detailed information of Materials and Methods.
Assuntos
Hipóxia/metabolismo , Mitocôndrias/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Doenças Retinianas/metabolismo , Animais , Movimento Celular , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Humanos , Hipóxia/complicações , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Processamento Associado a PTB/genética , Receptores de LDL/genética , Retina/metabolismo , Doenças Retinianas/etiologia , Doenças Retinianas/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Circular RNAs (circRNAs) represent an abundant and conserved entity of non-coding RNAs; however, the principles of biogenesis are currently not fully understood. Here, we identify two factors, splicing factor proline/glutamine rich (SFPQ) and non-POU domain-containing octamer-binding protein (NONO), to be enriched around circRNA loci. We observe a subclass of circRNAs, coined DALI circRNAs, with distal inverted Alu elements and long flanking introns to be highly deregulated upon SFPQ knockdown. Moreover, SFPQ depletion leads to increased intron retention with concomitant induction of cryptic splicing, premature transcription termination, and polyadenylation, particularly prevalent for long introns. Aberrant splicing in the upstream and downstream regions of circRNA producing exons are critical for shaping the circRNAome, and specifically, we identify missplicing in the immediate upstream region to be a conserved driver of circRNA biogenesis. Collectively, our data show that SFPQ plays an important role in maintaining intron integrity by ensuring accurate splicing of long introns, and disclose novel features governing Alu-independent circRNA production.
Assuntos
Íntrons , Fator de Processamento Associado a PTB/genética , Splicing de RNA , RNA Circular/metabolismo , Animais , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Fator de Processamento Associado a PTB/metabolismoRESUMO
RNA-binding proteins (RBPs) are a class of proteins known for their diverse roles in RNA biogenesis, from regulating transcriptional processes in the nucleus to facilitating translation in the cytoplasm. With higher demand for RNA metabolism in the nervous system, RBP misregulation has been linked to a wide range of neurological and neurodegenerative diseases. One of the emerging RBPs implicated in neuronal function and neurodegeneration is splicing factor proline- and glutamine-rich (SFPQ). SFPQ is a ubiquitous and abundant RBP that plays multiple regulatory roles in the nucleus such as paraspeckle formation, DNA damage repair, and various transcriptional regulation processes. An increasing number of studies have demonstrated the nuclear and also cytoplasmic roles of SFPQ in neurons, particularly in post-transcriptional regulation and RNA granule formation. Not surprisingly, the misregulation of SFPQ has been linked to pathological features shown by other neurodegenerative disease-associated RBPs such as aberrant RNA splicing, cytoplasmic mislocalization, and aggregation. In this review, we discuss recent findings on the roles of SFPQ with a particular focus on those in neuronal development and homeostasis as well as its implications in neurodegenerative diseases.
Assuntos
Doenças Neurodegenerativas/genética , Neurônios/metabolismo , Fator de Processamento Associado a PTB/genética , Splicing de RNA , RNA Mensageiro/genética , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Modelos Moleculares , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Fator de Processamento Associado a PTB/química , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismoRESUMO
The coupling of alternative splicing with the nonsense-mediated decay (NMD) pathway maintains quality control of the transcriptome in eukaryotes by eliminating transcripts with premature termination codons (PTC) and fine-tunes gene expression. Long noncoding RNA (lncRNA) can regulate multiple cellular processes, including alternative splicing. Previously, murine Morrbid (myeloid RNA repressor of Bcl2l11 induced death) lncRNA was described as a locus-specific controller of the lifespan of short-living myeloid cells via transcription regulation of the apoptosis-related Bcl2l11 protein. Here, we report that murine Morrbid lncRNA in hepatocytes participates in the regulation of proto-oncogene NRAS (neuroblastoma RAS viral oncogene homolog) splicing, including the formation of the isoform with PTC. We observed a significant increase of the NRAS isoform with PTC in hepatocytes with depleted Morrbid lncRNA. We demonstrated that the NRAS isoform with PTC is degraded via the NMD pathway. This transcript is presented almost only in the nucleus and has a half-life ~four times lower than other NRAS transcripts. Additionally, in UPF1 knockdown hepatocytes (the key NMD factor), we observed a significant increase of the NRAS isoform with PTC. By a modified capture hybridization (CHART) analysis of the protein targets, we uncovered interactions of Morrbid lncRNA with the SFPQ (splicing factor proline and glutamine rich)-NONO (non-POU domain-containing octamer-binding protein) splicing complex. Finally, we propose the regulation mechanism of NRAS splicing in murine hepatocytes by alternative splicing coupled with the NMD pathway with the input of Morrbid lncRNA.
Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a DNA/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Fator de Processamento Associado a PTB/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Animais , Códon sem Sentido/genética , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/metabolismo , Camundongos , Complexos Multiproteicos/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Transcriptoma/genéticaRESUMO
Increasing evidence indicates that long non-coding RNAs (lncRNAs) play vital roles in the tumorigenesis and progression of cancers. However, the functions and regulatory mechanisms of lncRNAs in nasopharyngeal carcinoma (NPC) are still largely unknown. Our previous lncRNA expression profiles identified that LINC01503 was overexpressed in NPC. Here, we verified that LINC01503 was highly expressed in NPC and correlated with poor prognosis. LINC01503 promoted NPC cell proliferation, migration, and invasion in vitro, and facilitated tumor growth and metastasis in vivo. Mechanistically, LINC01503 recruited splicing factor proline-and glutamine-rich (SFPQ) to activate Fos like 1 (FOSL1) transcription, and ectopic expression of FOSL1 reversed the suppressive effect of LINC01503 knockdown on NPC progression. Moreover, androgen receptor (AR)-mediated transcription activation was responsible for the overexpression of LINC01503, and AR ligand-dependent cell growth, migration, and invasion in NPC cells. Taken together, our findings reveal that AR-induced LINC01503 can promote NPC progression through the SFPQ-FOSL1 axis, which represents a novel prognostic biomarker and therapeutic target for NPC patients.
Assuntos
Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Fator de Processamento Associado a PTB/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Longo não Codificante/genética , Receptores Androgênicos/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/terapia , Fator de Processamento Associado a PTB/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
In recent years, a subgroup of B-cell precursor acute lymphoblastic leukemia (BCP ALL) without an established abnormality ("B-other") has been shown to be characterized by rearrangements of ABL1, ABL2, CSF1R, or PDGFRB (a.k.a. ABL-class genes). Using FISH with probes for these genes, we screened 55 pediatric and 50 adult B-other cases. Three (6%) of the adult but none of the childhood B-other cases were positive for ABL-class aberrations. RT-PCR and sequencing confirmed a rare SFPQ-ABL1 fusion in one adult B-other case with t(1;9)(p34;q34). Only six SFPQ-ABL1-positive BCP ALLs have been reported, present case included. A review of these shows that all harbored fusions between exon 9 of SFPQ and exon 4 of ABL1, that the fusion is typically found in adolescents/younger adults without hyperleukocytosis, and that IKZF1 deletions are recurrent. The few patients not treated with tyrosine kinase inhibitors (TKIs) and/or allogeneic stem cell transplantation relapsed, strengthening the notion that TKI should be added to the therapy of SFPQ-ABL1-positive BCP ALL.
Assuntos
Proteínas de Fusão Oncogênica/genética , Fator de Processamento Associado a PTB/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-abl/genética , Adolescente , Antineoplásicos/uso terapêutico , Criança , Pré-Escolar , Feminino , Humanos , Fator de Transcrição Ikaros/genética , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer, yet long-term treatment often causes acquired resistance, which results in recurrence and metastasis. Recent studies have revealed that RNA-binding proteins (RBP) are involved in tumorigenesis. Here, we demonstrate that PSF/SFPQ is an RBP that potentially predicts poor prognosis of patients with ER-positive breast cancer by posttranscriptionally regulating ERα (ESR1) mRNA expression. Strong PSF immunoreactivity correlated with shorter overall survival in patients with ER-positive breast cancer. PSF was predominantly expressed in a model of tamoxifen-resistant breast cancer cells, and depletion of PSF attenuated proliferation of cultured cells and xenografted tumors. PSF expression was significantly associated with estrogen signaling. PSF siRNA downregulated ESR1 mRNA by inhibiting nuclear export of the RNA. Integrative analyses of microarray and RNA immunoprecipitation sequencing also identified SCFD2, TRA2B, and ASPM as targets of PSF. Among the PSF targets, SCFD2 was a poor prognostic indicator of breast cancer and SCFD2 knockdown significantly suppressed breast cancer cell proliferation. Collectively, this study shows that PSF plays a pathophysiologic role in ER-positive breast cancer by posttranscriptionally regulating expression of its target genes such as ESR1 and SCFD2. Overall, PSF and SCFD2 could be potential diagnostic and therapeutic targets for primary and hormone-refractory breast cancers. SIGNIFICANCE: This study defines oncogenic roles of RNA-binding protein PSF, which exhibits posttranscriptional regulation in ER-positive breast cancer.