Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 51
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biochem Biophys Res Commun ; 562: 139-145, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34052659

RESUMO

We recently isolated a novel co-activator of peroxisome proliferator-activated receptor γ, helicase with zinc finger 2 (HELZ2). HELZ2 null mice were resistant to diet-induced obesity and NAFFL/NASH, and HELZ2 was phosphorylated at tyrosine residues. In order to find a factor related to HELZ2, we analyzed products co-immunoprecipitated with phosphorylated HELZ2 by mass spectrometry analyses. We identified proline- and glutamine-rich (SFPQ) as a protein associating with tyrosine-phosphorylated HELZ2. The knockdown of SFPQ in 3T3-L1 cells downregulated mRNA levels of transcription factors including Krox20, Cebpß, and Cebpδ: key factors for early-stage adipocyte differentiation. In addition, knockdown of SFPQ inhibited 3T3-L1 cell differentiation to mature adipocytes. These findings demonstrated that SFPQ associating with HELZ2 is an important novel transcriptional regulator of adipocyte differentiation.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , PPAR gama/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Animais , Regulação da Expressão Gênica , Células HeLa , Humanos , Gotículas Lipídicas/metabolismo , Camundongos , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo
2.
J Cell Sci ; 134(4)2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33495278

RESUMO

The expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteína C9orf72 , Expansão das Repetições de DNA , Fator de Processamento Associado a PTB/metabolismo , Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Dipeptídeos , Demência Frontotemporal/genética , Humanos , Mutação/genética , RNA
3.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33284322

RESUMO

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.


Assuntos
Transporte Axonal , Axônios/metabolismo , Fator de Processamento Associado a PTB/metabolismo , RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Grânulos Citoplasmáticos/metabolismo , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos , Mitocôndrias/metabolismo , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo
4.
Int J Mol Sci ; 21(19)2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-32998269

RESUMO

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/metabolismo
5.
Cell Rep ; 32(12): 108184, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32966782

RESUMO

Oncoproteins such as the BRAFV600E kinase endow cancer cells with malignant properties, but they also create unique vulnerabilities. Targeting of BRAFV600E-driven cytoplasmic signaling networks has proved ineffective, as patients regularly relapse with reactivation of the targeted pathways. We identify the nuclear protein SFPQ to be synthetically lethal with BRAFV600E in a loss-of-function shRNA screen. SFPQ depletion decreases proliferation and specifically induces S-phase arrest and apoptosis in BRAFV600E-driven colorectal and melanoma cells. Mechanistically, SFPQ loss in BRAF-mutant cancer cells triggers the Chk1-dependent replication checkpoint, results in decreased numbers and reduced activities of replication factories, and increases collision between replication and transcription. We find that BRAFV600E-mutant cancer cells and organoids are sensitive to combinations of Chk1 inhibitors and chemically induced replication stress, pointing toward future therapeutic approaches exploiting nuclear vulnerabilities induced by BRAFV600E.


Assuntos
Neoplasias Colorretais/genética , Mutação/genética , Fator de Processamento Associado a PTB/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Mutações Sintéticas Letais/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Neoplasias Colorretais/patologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Feminino , Humanos , Hidroxiureia/farmacologia , Camundongos Nus , Rad51 Recombinase/metabolismo , Reprodutibilidade dos Testes , Fase S/efeitos dos fármacos , Fase S/genética , Estresse Fisiológico/efeitos dos fármacos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
6.
Brain ; 143(8): 2398-2405, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32770214

RESUMO

Fused in sarcoma (FUS) is genetically and clinicopathologically linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). We have previously reported that intranuclear interactions of FUS and splicing factor, proline- and glutamine-rich (SFPQ) contribute to neuronal homeostasis. Disruption of the FUS-SFPQ interaction leads to an increase in the ratio of 4-repeat tau (4R-tau)/3-repeat tau (3R-tau), which manifests in FTLD-like phenotypes in mice. Here, we examined FUS-SFPQ interactions in 142 autopsied individuals with FUS-related ALS/FTLD (ALS/FTLD-FUS), TDP-43-related ALS/FTLD (ALS/FTLD-TDP), progressive supranuclear palsy, corticobasal degeneration, Alzheimer's disease, or Pick's disease as well as controls. Immunofluorescent imaging showed impaired intranuclear co-localization of FUS and SFPQ in neurons of ALS/FTLD-FUS, ALS/FTLD-TDP, progressive supranuclear palsy and corticobasal degeneration cases, but not in Alzheimer's disease or Pick's disease cases. Immunoprecipitation analyses of FUS and SFPQ revealed reduced interactions between the two proteins in ALS/FTLD-TDP and progressive supranuclear palsy cases, but not in those with Alzheimer disease. Furthermore, the ratio of 4R/3R-tau was elevated in cases with ALS/FTLD-TDP and progressive supranuclear palsy, but was largely unaffected in cases with Alzheimer disease. We concluded that impaired interactions between intranuclear FUS and SFPQ and the subsequent increase in the ratio of 4R/3R-tau constitute a common pathogenesis pathway in FTLD spectrum diseases.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Neurônios/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteinopatias TDP-43/metabolismo , Idoso , Esclerose Amiotrófica Lateral/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Degeneração Lobar Frontotemporal/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/patologia , Proteinopatias TDP-43/patologia , Proteínas tau/metabolismo
7.
Oncogene ; 39(34): 5616-5632, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661324

RESUMO

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/métodos
8.
Acta Neuropathol ; 140(3): 317-339, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32577828

RESUMO

Dysfunctional RNA-binding proteins (RBPs) have been implicated in several neurodegenerative disorders. Recently, this paradigm of RBPs has been extended to pathophysiology of Alzheimer's disease (AD). Here, we identified disease subtype specific variations in the RNA-binding proteome (RBPome) of sporadic AD (spAD), rapidly progressive AD (rpAD), and sporadic Creutzfeldt Jakob disease (sCJD), as well as control cases using RNA pull-down assay in combination with proteomics. We show that one of these identified proteins, splicing factor proline and glutamine rich (SFPQ), is downregulated in the post-mortem brains of rapidly progressive AD patients, sCJD patients and 3xTg mice brain at terminal stage of the disease. In contrast, the expression of SFPQ was elevated at early stage of the disease in the 3xTg mice, and in vitro after oxidative stress stimuli. Strikingly, in rpAD patients' brains SFPQ showed a significant dislocation from the nucleus and cytoplasmic colocalization with TIA-1. Furthermore, in rpAD brain lesions, SFPQ and p-tau showed extranuclear colocalization. Of note, association between SFPQ and tau-oligomers in rpAD brains suggests a possible role of SFPQ in oligomerization and subsequent misfolding of tau protein. In line with the findings from the human brain, our in vitro study showed that SFPQ is recruited into TIA-1-positive stress granules (SGs) after oxidative stress induction, and colocalizes with tau/p-tau in these granules, providing a possible mechanism of SFPQ dislocation through pathological SGs. Furthermore, the expression of human tau in vitro induced significant downregulation of SFPQ, suggesting a causal role of tau in the downregulation of SFPQ. The findings from the current study indicate that the dysregulation and dislocation of SFPQ, the subsequent DNA-related anomalies and aberrant dynamics of SGs in association with pathological tau represents a critical pathway which contributes to rapid progression of AD.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/patologia , Fator de Processamento Associado a PTB/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Citoplasma/metabolismo , Regulação para Baixo/fisiologia , Humanos , Camundongos Transgênicos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
9.
Cancer Res ; 80(11): 2230-2242, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32213542

RESUMO

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.


Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Fator de Processamento Associado a PTB/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Prognóstico , Processamento Pós-Transcricional do RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Tamoxifeno/farmacologia
10.
Cancer Med ; 9(5): 1855-1866, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31953923

RESUMO

Long non-coding RNA (lncRNA) is emerging as a pivotal regulator in tumorigenesis and aggressive progression. Here, we focused on an oncogenic lncRNA, ARAP1 antisense RNA 1 (ARAP1-AS1), which was notably upregulated in cervical cancer (CC) tissues, cell lines and serum. High ARAP1-AS1 expression was closely associated with larger tumor size, advanced FIGO stage as well as lymph node metastasis. Importantly, it was identified as an effective diagnostic and prognostic biomarker for CC. In vitro and in vivo assays showed that knockdown of ARAP1-AS1 inhibited, while overexpression of ARAP1-AS1 promoted CC cell growth and dissemination. Stepwise mechanistic dissection unveiled that ARAP1-AS1 could directly interact with PSF to release PTB, resulting in accelerating the internal ribosome entry site (IRES)-driven translation of proto-oncogene c-Myc, thereby facilitating CC development and progression. Moreover, c-Myc was able to transcriptionally activate ARAP1-AS1 by directly binding to the E-box motif located on ARAP1-AS1 promoter. Taken together, our findings clearly reveal the crucial role of ARAP1-AS1 in CC tumorigenesis and metastasis via regulation of c-Myc translation, targeting ARAP1-AS1 and its related regulatory loop implicates the therapeutic possibility for CC patients.


Assuntos
Carcinogênese/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/metabolismo , Neoplasias do Colo do Útero/genética , Animais , Linhagem Celular Tumoral , Elementos E-Box/genética , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sítios Internos de Entrada Ribossomal/genética , Neoplasias Pulmonares/secundário , Camundongos , Fator de Processamento Associado a PTB/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , Ativação Transcricional , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cell Chem Biol ; 27(3): 283-291.e6, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31981477

RESUMO

The fat mass and obesity-associated protein (FTO) is the first identified demethylase of the internal RNA modification N6-methyladenosine (m6A), which also exhibits demethylation activity toward N6,2'-O-dimethyladenosine (m6Am) and N1-methyladenosine (m1A). Demethylation of m6A at specific sites on target transcripts is a key enzymatic function of FTO that modulates diverse physiological and/or pathological processes. However, how FTO selects target RNA and whether additional interaction proteins facilitate this process remain elusive. Herein, via the genetically encoded and site-specific photocrosslinking strategy, we identified the major RNA-binding protein SFPQ as a direct interaction partner of FTO. Our study showed that FTO and SFPQ were located in close proximity throughout the transcriptome and that overexpression of SFPQ led to the demethylation of adjacent m6As, likely through recruiting FTO to these specific RNA sites. These results uncovered a new layer of regulation mechanism that may assist FTO to gain substrate specificity.


Assuntos
Fator de Processamento Associado a PTB/metabolismo , RNA/metabolismo , Desmetilação , Células HEK293 , Humanos , Fator de Processamento Associado a PTB/química , RNA/química , Especificidade por Substrato
12.
Reproduction ; 158(3): 267-280, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299635

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células Intersticiais do Testículo/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Masculino , Camundongos , Fator de Processamento Associado a PTB/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Células de Sertoli/metabolismo , Testículo/crescimento & desenvolvimento
13.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 439-449, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204691

RESUMO

Splicing factor proline/glutamine-rich (SFPQ) is an essential RNA-binding protein that is implicated in many aspects of nuclear function. The structures of SFPQ and two paralogs, non-POU domain-containing octamer-binding protein and paraspeckle component 1, from the Drosophila behavior human splicing protein family have previously been characterized. The unusual arrangement of the four domains, two RNA-recognition motifs (RRMs), a conserved region termed the NonA/paraspeckle (NOPS) domain and a C-terminal coiled coil, in the intertwined dimer provides a potentially unique RNA-binding surface. However, the molecular details of how the four RRMs in the dimeric SFPQ interact with RNA remain to be characterized. Here, a new crystal structure of the dimerization domain of human SFPQ in the C-centered orthorhombic space group C2221 with one monomer in the asymmetric unit is presented. Comparison of the new crystal structure with the previously reported structure of SFPQ and analysis of the solution small-angle X-scattering data revealed subtle domain movements in the dimerization domain of SFPQ, supporting the concept of multiple conformations of SFPQ in equilibrium in solution. The domain movement of RRM1, in particular, may reflect the complexity of the RNA substrates of SFPQ. Taken together, the crystal and solution structure analyses provide a molecular basis for further investigation into the plasticity of nucleic acid binding by SFPQ in the absence of the structure in complex with its cognate RNA-binding partners.


Assuntos
Fator de Processamento Associado a PTB/química , Fator de Processamento Associado a PTB/metabolismo , Multimerização Proteica , RNA/metabolismo , Espalhamento a Baixo Ângulo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , RNA/química
14.
RNA Biol ; 16(7): 940-949, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30951404

RESUMO

SLC47A2 encodes MATE 2-K in the kidney, which mediates the secretion of certain endogenous and exogenous compounds. SLC47A2 was dramatically repressed in patients with renal cell carcinoma (RCC), and a lower level of SLC47A2 might act as a negative prognostic marker, although the mechanism is not well understood. In this study, we aimed to investigate the mechanism via which SLC47A2 is downregulated in RCC. Based on the annotation information of the SLC47A2 locus available in the UCSC genome browser database, we identified a novel lncRNA, which is transcribed from the SLC47A2 locus and named it SANT1. Overexpression and knock-down assays were performed to investigate the effects of SANT1 on cis-regulation of SLC47A2. We verified the direct binding between SANT1 and SFPQ/E2F1/HDAC1 using the cross-linking and immunoprecipitation (CLIP) assay. Chromatin immunoprecipitation was performed to confirm the molecular mechanism via which SANT1 activates the transcription of the SLC47A2 coding region. We observed that SANT1 can cis-regulate its own genetic locus. In tumour-adjacent tissues, the SLC47A2 locus highly expresses SANT1, which can remove the regulatory SFPQ/E2F1/HDAC1 suppressor complex from the promoter region, thereby significantly increasing the levels of the H3K27ac modification and RNAPII binding. Owing to a low SANT1 level, the binding of this inhibitory complex in the promoter region is upregulated in RCC, which results in silencing of the SLC47A2 coding region. In conclusion, we identified a novel lncRNA and elucidated the mechanism via which it regulates SLC47A2 expression in RCC.


Assuntos
Carcinoma de Células Renais/genética , Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 1/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias Renais/genética , Modelos Biológicos , Conformação de Ácido Nucleico , Proteínas de Transporte de Cátions Orgânicos/genética , Ligação Proteica , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genética
15.
Cell Mol Life Sci ; 76(11): 2043-2058, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980111

RESUMO

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.


Assuntos
Código Genético , MicroRNAs/metabolismo , Repetições de Microssatélites , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Sítios de Ligação , Genoma Humano , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , MicroRNAs/genética , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Ligação Proteica , RNA Mensageiro/genética
16.
Nucleic Acids Res ; 47(10): 5381-5394, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30931476

RESUMO

Alternative splicing is facilitated by accessory proteins that guide spliceosome subunits to the primary transcript. Many of these splicing factors recognize the RNA polymerase II tail, but SFPQ is a notable exception even though essential for mammalian RNA processing. This study reveals a novel role for Dido3, one of three Dido gene products, in alternative splicing. Binding of the Dido3 amino terminus to histones and to the polymerase jaw domain was previously reported, and here we show interaction between its carboxy terminus and SFPQ. We generated a mutant that eliminates Dido3 but preserves other Dido gene products, mimicking reduced Dido3 levels in myeloid neoplasms. Dido mutation suppressed SFPQ binding to RNA and increased skipping for a large group of exons. Exons bearing recognition sequences for alternative splicing factors were nonetheless included more efficiently. Reduced SFPQ recruitment may thus account for increased skipping of SFPQ-dependent exons, but could also generate a splicing factor surplus that becomes available to competing splice sites. Taken together, our data indicate that Dido3 is an adaptor that controls SFPQ utilization in RNA splicing. Distributing splicing factor recruitment over parallel pathways provides mammals with a simple mechanism to regulate exon usage while maintaining RNA splicing efficiency.


Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Fator de Processamento Associado a PTB/metabolismo , Animais , Reagentes para Ligações Cruzadas/química , Éxons , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Mutação , Ligação Proteica , RNA/química , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Spliceossomos/metabolismo
17.
Nat Commun ; 10(1): 1001, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824709

RESUMO

In vertebrates, the telomere repeat containing long, non-coding RNA TERRA is prone to form RNA:DNA hybrids at telomeres. This results in the formation of R-loop structures, replication stress and telomere instability, but also contributes to alternative lengthening of telomeres (ALT). Here, we identify the TERRA binding proteins NONO and SFPQ as novel regulators of RNA:DNA hybrid related telomere instability. NONO and SFPQ locate at telomeres and have a common role in suppressing RNA:DNA hybrids and replication defects at telomeres. NONO and SFPQ act as heterodimers to suppress fragility and homologous recombination at telomeres, respectively. Combining increased telomere fragility with unleashing telomere recombination upon NONO/SFPQ loss of function causes massive recombination events, involving 35% of telomeres in ALT cells. Our data identify the RNA binding proteins SFPQ and NONO as novel regulators at telomeres that collaborate to ensure telomere integrity by suppressing telomere fragility and homologous recombination triggered by RNA:DNA hybrids.


Assuntos
DNA/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Hibridização de Ácido Nucleico , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Telômero/metabolismo , Animais , Linhagem Celular Tumoral , Replicação do DNA , Proteínas de Ligação a DNA , Recombinação Homóloga , Humanos , Camundongos , RNA não Traduzido , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo
18.
J Cell Sci ; 132(5)2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30745340

RESUMO

The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Demência Frontotemporal/genética , Neurônios Motores/fisiologia , Complexos Multiproteicos/metabolismo , Mutação/genética , RNA Nuclear/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Animais , Proteína C9orf72/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espaço Intranuclear , Camundongos , Fator de Processamento Associado a PTB/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Nuclear/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos
19.
Cell Mol Life Sci ; 76(10): 2015-2030, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30725116

RESUMO

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.


Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteínas de Ligação a DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteínas Associadas à Matriz Nuclear/genética , Fatores de Transcrição de Octâmero/genética , Fator de Processamento Associado a PTB/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Interferência de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
20.
Biochem Biophys Res Commun ; 510(1): 65-71, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30661786

RESUMO

The encephalomyocarditis virus (EMCV) is a single-stranded RNA virus that induces sudden death, diabetes, myocarditis and nervous disorders in non-human primates. The rapid development of xenografts such as heart transplantation from pig to human raises the issue of EMCV safety in human cells. SFPQ, a proline and glutamine rich splicing factor that participates in diverse molecular functions including paraspeckle formation, microRNA synthesis and transcription regulation, is known to regulate host innate immune response to viruses. However, the role of SFPQ in EMCV infection remains unclear. Here we reported that the SFPQ was essential for EMCV replication. Depletion of SFPQ impaired EMCV production, while forced expression of SFPQ promoted viral replication. Mechanistically, loss of SFPQ affected the transcription profile of host mitochondria pathway related genes. In addition, cellular SFPQ was exploited by EMCV and accumulated in cytoplasm and it interacted with eukaryotic initiation factors and ribosomal proteins to facilitate internal ribosome entry site (IRES)-dependent translation of EMCV protein. Altogether, our work discovered host SFPQ as a new target to inhibit EMCV replication and infection.


Assuntos
Vírus da Encefalomiocardite/fisiologia , Fator de Processamento Associado a PTB/metabolismo , Replicação Viral , Infecções por Cardiovirus , Humanos , Sítios Internos de Entrada Ribossomal , Fator de Processamento Associado a PTB/fisiologia , Proteínas Virais/biossíntese
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...