RESUMEN
The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway.
Asunto(s)
ADN/inmunología , Herpesvirus Humano 8/inmunología , Inmunidad Innata , ARN Largo no Codificante/inmunología , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/virología , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/inmunología , Autoantígeno Ku/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/inmunología , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/inmunología , Factor de Empalme Asociado a PTB/metabolismo , Unión Proteica , Interferencia de ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Factores de Transcripción , TransfecciónRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Ciclinas/genética , Demencia Frontotemporal/genética , Factor de Empalme Asociado a PTB/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Células HEK293 , Humanos , Agregado de Proteínas/genética , Mapas de Interacción de Proteínas/genética , Proteolisis , ARN/genética , ARN/metabolismo , Procesamiento Postranscripcional del ARN/genética , Proteínas de Unión al ARN/genéticaRESUMEN
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.
Asunto(s)
ARN Largo no Codificante , Proteínas de Unión al ARN , Animales , Humanos , Ratones , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/metabolismo , Empalme del ARN , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
SFPQ is a ubiquitous nuclear RNA-binding protein implicated in many aspects of RNA biogenesis. Importantly, nuclear depletion and cytoplasmic accumulation of SFPQ has been linked to neuropathological conditions such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Here, we describe a molecular mechanism by which SFPQ is mislocalized to the cytoplasm. We report an unexpected discovery of the infinite polymerization of SFPQ that is induced by zinc binding to the protein. The crystal structure of human SFPQ in complex with zinc at 1.94 Å resolution reveals intermolecular interactions between SFPQ molecules that are mediated by zinc. As anticipated from the crystal structure, the application of zinc to primary cortical neurons induced the cytoplasmic accumulation and aggregation of SFPQ. Mutagenesis of the three zinc-coordinating histidine residues resulted in a significant reduction in the zinc-binding affinity of SFPQ in solution and the zinc-induced cytoplasmic aggregation of SFPQ in cultured neurons. Taken together, we propose that dysregulation of zinc availability and/or localization in neuronal cells may represent a mechanism for the imbalance in the nucleocytoplasmic distribution of SFPQ, which is an emerging hallmark of neurodegenerative diseases including AD and ALS.
Asunto(s)
Neuronas/metabolismo , Factor de Empalme Asociado a PTB/ultraestructura , Proteínas de Unión al ARN/ultraestructura , ARN/genética , Enfermedad de Alzheimer/genética , Esclerosis Amiotrófica Lateral/genética , Núcleo Celular/genética , Cristalografía por Rayos X , Citoplasma/genética , Humanos , Neuronas/patología , Factor de Empalme Asociado a PTB/química , Factor de Empalme Asociado a PTB/genética , Polimerizacion , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Zinc/metabolismoRESUMEN
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.
Asunto(s)
Hipoxia/metabolismo , Mitocondrias/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , Enfermedades de la Retina/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Humanos , Hipoxia/complicaciones , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Empalme Asociado a PTB/genética , Receptores de LDL/genética , Retina/metabolismo , Enfermedades de la Retina/etiología , Enfermedades de la Retina/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
DNA hemicatenanes (HCs) are four-way junctions in which one strand of a double-stranded helix is catenated with one strand of another double-stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, we sought to purify proteins capable of binding specifically HCs by fractionating nuclear extracts from HeLa cells. This approach identified three RNA-binding proteins: the Tudor-staphylococcal nuclease domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the paraspeckle protein component 1 and the splicing factor proline- and glutamine-rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in Escherichia coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited specificity for HCs, opening the interesting possibility of a link between the basic transcription machinery and HC structures via SND1.
Asunto(s)
Catenanos/metabolismo , ADN/genética , Endonucleasas/genética , Transcripción Genética , Animales , Catenanos/química , Cromosomas/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/metabolismo , Células HeLa , Humanos , Factor de Empalme Asociado a PTB/genética , Unión Proteica/genética , Proteínas de Unión al ARN/genética , Recombinación Genética/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Fusión Oncogénica/genética , Factor de Empalme Asociado a PTB/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-abl/genética , Adolescente , Antineoplásicos/uso terapéutico , Niño , Preescolar , Femenino , Humanos , Factor de Transcripción Ikaros/genética , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Amyloid precursor protein (APP) is a representative gene related to Alzheimer's disease (AD). Androgens function by binding to the androgen receptor (AR). Both androgen and RNA-binding protein PSF play a role in the pathology of AD. However, the involvement of AR and PSF in APP regulation in neuron has not been investigated. Here, we explored the regulatory mechanism of APP expression by AR and PSF using neuron-derived cells. We demonstrated that androgen up-regulates the production of APP at the mRNA and protein levels. This induction is enhanced by AR over-expression and inhibited by its silencing. One candidate AR-binding region was identified in the intron region of APP and validated its activity as AR-dependent enhancer by the luciferase assay. Furthermore, the public transcriptome data of brain tissues of mice indicated that APP is regulated by PSF post-transcriptionally. We observed a decreased expression of APP after PSF knockdown and interaction of PSF with the APP transcript. Moreover, we revealed that silencing of PSF inhibited the stability of the APP mRNA. Thus, these results presented a new regulatory mechanism of APP expression by androgen through AR-mediated transcription and PSF at the post-transcriptional level that might be associated with the occurrence of AD.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Andrógenos/genética , Andrógenos/metabolismo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Factor de Empalme Asociado a PTB/genética , Unión Proteica , ARN Mensajero/metabolismo , Receptores Androgénicos , Transcriptoma , Regulación hacia ArribaRESUMEN
Protein production, genomic RNA replication, and virion assembly during infection by picornaviruses like human rhinovirus and poliovirus take place in the cytoplasm of infected human cells, making them the quintessential cytoplasmic pathogens. However, a growing body of evidence suggests that picornavirus replication is promoted by a number of host proteins localized normally within the host cell nucleus. To systematically identify such nuclear proteins, we focused on those that appear to re-equilibrate from the nucleus to the cytoplasm during infection of HeLa cells with human rhinovirus via quantitative protein mass spectrometry. Our analysis revealed a highly selective re-equilibration of proteins with known mRNA splicing and transport-related functions over nuclear proteins of all other functional classes. The multifunctional splicing factor proline and glutamine rich (SFPQ) was identified as one such protein. We found that SFPQ is targeted for proteolysis within the nucleus by viral proteinase 3CD/3C, and a fragment of SFPQ was shown to migrate to the cytoplasm at mid-to-late times of infection. Cells knocked down for SFPQ expression showed significantly reduced rhinovirus titers, viral protein production, and viral RNA accumulation, consistent with SFPQ being a pro-viral factor. The SFPQ fragment that moved into the cytoplasm was able to bind rhinovirus RNA either directly or indirectly. We propose that the truncated form of SFPQ promotes viral RNA stability or replication, or virion morphogenesis. More broadly, our findings reveal dramatic changes in protein compartmentalization during human rhinovirus infection, allowing the virus to systematically hijack the functions of proteins not normally found at its cytoplasmic site of replication.
Asunto(s)
Núcleo Celular/fisiología , Citoplasma/virología , Interacciones Huésped-Patógeno , Rhinovirus/fisiología , Transporte Activo de Núcleo Celular , Citoplasma/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/metabolismo , Transporte de Proteínas , Proteolisis , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20 µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.
Asunto(s)
Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Bencimidazoles/farmacología , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Proteínas de Unión al ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas Asociadas a Matriz Nuclear/genética , Factores de Transcripción de Octámeros/genética , Factor de Empalme Asociado a PTB/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Interferencia de ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
MicroRNAs are small endogenous RNAs that pair and bind to sites on mRNAs to direct post-transcriptional repression. However, there is a possibility that microRNAs directly influence protein structure and activity, and this influence can be termed post-translational riboregulation. This conceptual review explores the literature on neurodegenerative disorders. Research on the association between neurodegeneration and RNA-repeat toxicity provides data that support a protein-RNA recognition code. For example, this code explains why hnRNP H and SFPQ proteins, which are involved in amyotrophic lateral sclerosis, are sequestered by the (GGGGCC)n repeat sequence. Similarly, it explains why MNBL proteins and (CTG)n repeats in RNA, which are involved in myotonic dystrophy, are sequestered into RNA foci. Using this code, proteins involved in diseases can be identified. A simple protein BLAST search of the human genome for amino acid repeats that correspond to the nucleotide repeats reveals new proteins among already known proteins that are involved in diseases. For example, the (CAG)n repeat sequence, when transcribed into possible peptide sequences, leads to the identification of PTCD3, Rem2, MESP2, SYPL2, WDR33, COL23A1, and others. After confirming this approach on RNA repeats, in the next step, the code was used in the opposite manner. Proteins that are involved in diseases were compared with microRNAs involved in those diseases. For example, a reasonable correspondence of microRNA 9 and 107 with amyloid-ß-peptide (Aß42) was identified. In the last step, a miRBase search for micro-nucleotides, obtained by transcription of a prion amino acid sequence, revealed new microRNAs and microRNAs that have previously been identified as involved in prion diseases. This concept provides a useful key for designing RNA or peptide probes.
Asunto(s)
Código Genético , MicroARNs/metabolismo , Repeticiones de Microsatélite , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Sitios de Unión , Genoma Humano , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , MicroARNs/genética , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Distrofia Miotónica/patología , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Unión Proteica , ARN Mensajero/genéticaRESUMEN
We reported previously that a 2' fluoro-modified (2' F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5-10-5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2'-O-(2-methoxyethyl) (2' MOE) or 2',4'-constrained 2'-O-ethyl (cEt) did. Rapid degradation of these proteins and cytotoxicity were observed in cells treated with 2' F PS-ASO. Here, we report that 2' F gapmer PS-ASOs of different sequences caused reduction in levels of DBHS proteins and hepatotoxicity in mice. 2' F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were observed as early as 24 to 48 hours after a single administration of the 2' F PS-ASO. The observed effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2' F nucleoside metabolites. Instead, we found that 2' F PS-ASO associated with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates positively with the 2' F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2' F PS-ASO in vivo.
Asunto(s)
Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos Fosforotioatos/metabolismo , Transcriptoma/efectos de los fármacos , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos BALB C , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/metabolismo , Oligonucleótidos Fosforotioatos/genética , Unión Proteica/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcriptoma/genéticaRESUMEN
Developing therapeutic approaches are necessary for treating hormone-refractory prostate cancer. Activation of androgen receptor (AR) and its variants' expression along with the downstream signals are mostly important for disease progression. However, the mechanism for marked increases of AR signals and its expression is still unclear. Here, we revealed that various spliceosome genes are aberrantly induced by RNA-binding protein PSF, leading to enhancement of the splicing activities for AR expression. Our high-speed sequence analyses identified global PSF-binding transcripts. PSF was shown to stabilize and activate key long noncoding RNAs and AR-regulated gene expressions in prostate cancer cells. Interestingly, mRNAs of spliceosome-related genes are putative primary targets of PSF. Their gene expressions are up-regulated by PSF in hormone-refractory prostate cancer. Moreover, PSF coordinated these spliceosome proteins to form a complex to promote AR splicing and expression. Thus, targeting PSF and its related pathways implicates the therapeutic possibility for hormone-refractory prostate cancer.
Asunto(s)
Factor de Empalme Asociado a PTB/biosíntesis , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , ARN Largo no Codificante/genética , Receptores Androgénicos/biosíntesis , Empalmosomas/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Factor de Empalme Asociado a PTB/genética , Neoplasias de la Próstata Resistentes a la Castración/terapia , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptores Androgénicos/genética , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Empalme Alternativo/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al GTP Monoméricas/genética , Factor de Empalme Asociado a PTB/genética , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Animales , Codón sin Sentido/genética , Regulación del Desarrollo de la Expresión Génica , Hepatocitos/metabolismo , Ratones , Complejos Multiproteicos/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Transcriptoma/genéticaRESUMEN
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.
Asunto(s)
Enfermedades Neurodegenerativas/genética , Neuronas/metabolismo , Factor de Empalme Asociado a PTB/genética , Empalme del ARN , ARN Mensajero/genética , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Modelos Moleculares , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/citología , Factor de Empalme Asociado a PTB/química , Factor de Empalme Asociado a PTB/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/metabolismoRESUMEN
Expression profiles and subcellular localisations of core Drosophila behaviour/human splicing (DBHS) proteins (PSPC1, SFPQ and NONO) and NEAT1, a long noncoding RNA (lncRNA), are investigated in developing and adult mouse testes. Core DBHS proteins are markers for the distinct subnuclear domain termed paraspeckles, while a long NEAT1 isoform scaffold facilitates paraspeckle nucleation. Paraspeckles contain many proteins (>40) and are broadly involved in RNA metabolism, including transcriptional regulation by protein sequestration, nuclear retention of A-to-I edited RNA transcripts to regulate translation and promoting survival during cellular stress. Immunohistochemistry reveals cell-specific profiles for core DBHS paraspeckle protein expression, indicating their functional diversity. PSPC1 is an androgen receptor co-activator, and it is detected in differentiating Sertoli cell nuclei from day 15 onwards, as they develop androgen responsiveness. PSPC1 is nuclear in the most mature male germ cell type present at each age, from foetal to adult life. In adult mouse testes, PSPC1 and SFPQ are present in Sertoli cells, spermatocytes and round spermatids, while the NEAT1 lncRNA appears in the punctate nuclear foci delineating paraspeckles only within Leydig cells. Identification of NEAT1 in the cytoplasm of spermatogonia and spermatocytes must reflect non-paraspeckle-related functions. NONO was absent from germ cells but nuclear in Sertoli cells. Reciprocal nuclear profiles of PSPC1 and γ-H2AX in spermatogenic cells suggest that each performs developmentally regulated roles in stress responses. These findings demonstrate paraspeckles and paraspeckle-related proteins contribute to diverse functions during testis development and spermatogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Células Intersticiales del Testículo/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , Proteínas de Unión al ARN/metabolismo , Espermatogénesis/fisiología , Testículo/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Masculino , Ratones , Factor de Empalme Asociado a PTB/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Células de Sertoli/metabolismo , Testículo/crecimiento & desarrolloRESUMEN
Antisense oligonucleotides (ASOs) are versatile tools that can regulate multiple steps of RNA biogenesis in cells and living organisms. Significant improvements in delivery, potency, and stability have been achieved through modifications within the oligonucleotide backbone, sugar and heterocycles. However, these modifications can profoundly affect interactions between ASOs and intracellular proteins in ways that are only beginning to be understood. Here, we report that ASOs with specific backbone and sugar modifications can become localized to cytoplasmic ribonucleoprotein granules such as stress granules and those seeded by the aggregation of specific ASO-binding proteins such as FUS/TLS (FUS) and PSF/SFPQ (PSF). Further investigation into the basis for ASO-FUS binding illustrated the importance of ASO backbone and hydrophobic 2' sugar modifications and revealed that the C-terminal region of FUS is sufficient to retain ASOs in cellular foci. Taken together, the results of this study demonstrate that affinities of various nucleic acid binding domains for ASO depend on chemical modifications and further demonstrate how ASO-protein interactions influence the localization of ASOs.
Asunto(s)
Oligonucleótidos Antisentido/análisis , Factor de Empalme Asociado a PTB/metabolismo , Oligonucleótidos Fosforotioatos/análisis , Proteína FUS de Unión a ARN/metabolismo , Línea Celular , Núcleo Celular/química , Gránulos Citoplasmáticos/química , Humanos , Mutación , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , Factor de Empalme Asociado a PTB/genética , Oligonucleótidos Fosforotioatos/química , Oligonucleótidos Fosforotioatos/metabolismo , Agregado de Proteínas , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína FUS de Unión a ARN/análisis , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genéticaRESUMEN
The 5' UTR of Coxsackievirus B3 (CVB3) contains internal ribosome entry site (IRES), which allows cap-independent translation of the viral RNA and a 5'-terminal cloverleaf structure that regulates viral replication, translation and stability. Here, we demonstrate that host protein PSF (PTB associated splicing factor) interacts with the cloverleaf RNA as well as the IRES element. PSF was found to be an important IRES trans acting factor (ITAF) for efficient translation of CVB3 RNA. Interestingly, cytoplasmic abundance of PSF protein increased during CVB3 infection and this is regulated by phosphorylation status at two different amino acid positions. Further, PSF protein was up-regulated in CVB3 infection. The expression of CVB3-2A protease alone could also induce increased PSF protein levels. Furthermore, we observed the presence of an IRES element in the 5'UTR of PSF mRNA, which is activated during CVB3 infection and might contribute to the elevated levels of PSF. It appears that PSF IRES is also positively regulated by PTB, which is known to regulate CVB3 IRES. Taken together, the results suggest for the first time a novel mechanism of regulations of ITAFs during viral infection, where an ITAF undergoes IRES mediated translation, sustaining its protein levels under condition of translation shut-off.
Asunto(s)
Enterovirus Humano B/genética , Interacciones Huésped-Patógeno , Factor de Empalme Asociado a PTB/genética , Biosíntesis de Proteínas , ARN Viral/genética , Ribosomas/genética , Regiones no Traducidas 5' , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Enterovirus Humano B/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Sitios Internos de Entrada al Ribosoma , Conformación de Ácido Nucleico , Factor de Empalme Asociado a PTB/antagonistas & inhibidores , Factor de Empalme Asociado a PTB/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Ribosomas/metabolismo , Transducción de Señal , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
We here report a case of a distinct subtype of pigmented/melanocytic malignant PEComa with TFE3-SFPQ(PSF) rearrangement. The tumor involved the iliac region and clinically mimicked metastatic melanoma. The immunohistochemical assessment was supplemented with molecular studies including fluorescence in situ hybridization (FISH) and next-generation sequencing sarcoma panel (NGS). We also discuss the differential diagnosis of intraabdominal PEComas and emphasise the recent molecular reports on the TFE3 rearranged tumors.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Reordenamiento Génico , Melanoma/genética , Factor de Empalme Asociado a PTB/genética , Neoplasias de Células Epitelioides Perivasculares/genética , Sarcoma/genética , Biomarcadores de Tumor/genética , Humanos , Hibridación Fluorescente in SituRESUMEN
Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing.