RESUMO
Changes in the location of γ-tubulin ensure cell survival and preserve genome integrity. We investigated whether the nuclear accumulation of γ-tubulin facilitates the transport of proliferating cell nuclear antigen (PCNA) between the cytosolic and the nuclear compartment in mammalian cells. We found that the γ-tubulin meshwork assists in the recruitment of PCNA to chromatin. Also, decreased levels of γ-tubulin reduce the nuclear pool of PCNA. In addition, the γ-tubulin C terminus encodes a PCNA-interacting peptide (PIP) motif, and a γ-tubulin-PIP-mutant affects the nuclear accumulation of PCNA. In a cell-free system, PCNA and γ-tubulin formed a complex. In tumors, there is a significant positive correlation between TUBG1 and PCNA expression. Thus, we report a novel mechanism that constitutes the basis for tumor growth by which the γ-tubulin meshwork maintains indefinite proliferation by acting as an opportune scaffold for the transport of PCNA from the cytosol to the chromatin.
Assuntos
Cromatina/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Tubulina (Proteína)/fisiologia , Ciclo Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Humanos , Transporte Proteico , Origem de ReplicaçãoRESUMO
The RBP associated with lethal yellow mutation (RALY) is a member of the heterogeneous nuclear ribonucleoprotein family whose transcriptome and interactome have been recently characterized. RALY binds poly-U rich elements within several RNAs and regulates the expression as well as the stability of specific transcripts. Here we show that RALY binds PRMT1 mRNA and regulates its expression. PRMT1 catalyzes the arginine methylation of Fused in Sarcoma (FUS), an RNA-binding protein that interacts with RALY. We demonstrate that RALY down-regulation decreases protein arginine N-methyltransferase 1 levels, thus reducing FUS methylation. It is known that mutations in the FUS nuclear localization signal (NLS) retain the protein to the cytosol, promote aggregate formation, and are associated with amyotrophic lateral sclerosis. Confirming that inhibiting FUS methylation increases its nuclear import, we report that RALY knockout enhances FUS NLS mutants' nuclear translocation, hence decreasing aggregate formation. Furthermore, we characterize the RNA-dependent interaction of RALY with FUS in motor neurons. We show that mutations in FUS NLS as well as in RALY NLS reciprocally alter their localization and interaction with target mRNAs. These data indicate that RALY's activity is impaired in FUS pathology models, raising the possibility that RALY might modulate disease onset and/or progression.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neurônios Motores/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína FUS de Ligação a RNA/genética , Proteínas Repressoras/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Linhagem Celular Tumoral , Embrião de Mamíferos , Regulação da Expressão Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Camundongos , Neurônios Motores/citologia , Mutação , Sinais de Localização Nuclear , Cultura Primária de Células , Transporte Proteico , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
The heterogeneous nuclear ribonucleoproteins (hnRNP) form a large family of RNA-binding proteins that exert numerous functions in RNA metabolism. RALY is a member of the hnRNP family that binds poly-U-rich elements within several RNAs and regulates the expression of specific transcripts. RALY is up-regulated in different types of cancer, and its down-regulation impairs cell cycle progression. However, the RALY's role in regulating RNA levels remains elusive. Here, we show that numerous genes coding for factors involved in transcription and cell cycle regulation exhibit an altered expression in RALY-down-regulated HeLa cells, consequently causing impairments in transcription, cell proliferation, and cell cycle progression. Interestingly, by comparing the list of RALY targets with the list of genes affected by RALY down-regulation, we found an enrichment of RALY mRNA targets in the down-regulated genes upon RALY silencing. The affected genes include the E2F transcription factor family. Given its role as proliferation-promoting transcription factor, we focused on E2F1. We demonstrate that E2F1 mRNA stability and E2F1 protein levels are reduced in cells lacking RALY expression. Finally, we also show that RALY interacts with transcriptionally active chromatin in both an RNA-dependent and -independent manner and that this association is abolished in the absence of active transcription. Taken together, our results highlight the importance of RALY as an indirect regulator of transcription and cell cycle progression through the regulation of specific mRNA targets, thus strengthening the possibility of a direct gene expression regulation exerted by RALY.
Assuntos
Proliferação de Células/fisiologia , Fator de Transcrição E2F1/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/fisiologia , Transcrição Gênica/fisiologia , Ciclo Celular/genética , Fator de Transcrição E2F1/genética , Inativação Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Ligação Proteica , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica/genética , TranscriptomaRESUMO
We report a comprehensive study of the biocompatibility and neurocompatibility of titanium dioxide films (TiO2) prepared by Pulsed Microplasma Cluster Source (PMCS). This technique uses supersonic pulsed beams seeded by clusters of the metal oxide synthesized in a plasma discharge. The final stoichiometry of the TiO2 thin films is tuned changing the gas mixture, achieving stoichiometric or oxygen overstoichiometric films. All the films showed consistent biocompatibility and a spontaneous absorption of poly-d-lysine (PDL) that favors the adhesion and growth of murine cortical neurons. Moreover, the bioelectrical activity of the neuronal culture grown on the TiO2 film can be modulated by changing the chemistry of the surface. This work paves the way to develop a bio-hybrid neuromorphic device, where viable nerve cells are grown directly over a titanium dioxide film showing a network of memristors.
Assuntos
Materiais Biocompatíveis/química , Titânio/química , Potenciais de Ação/efeitos dos fármacos , Adsorção , Animais , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células HeLa , Humanos , Células MCF-7 , Camundongos , Microscopia de Força Atômica , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Polilisina/química , Polilisina/metabolismo , Propriedades de SuperfícieRESUMO
RALY is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP), a large family of RNA-binding proteins involved in many aspects of RNA metabolism. Although RALY interactome has been recently characterized, a comprehensive global analysis of RALY-associated RNAs is lacking and the biological function of RALY remains elusive. Here, we performed RIP-seq analysis to identify RALY interacting RNAs and assessed the role of RALY in gene expression. We demonstrate that RALY binds specific coding and non-coding RNAs and associates with translating mRNAs of mammalian cells. Among the identified transcripts, we focused on ANXA1 and H1FX mRNAs, encoding for Annexin A1 and for the linker variant of the histone H1X, respectively. Both proteins are differentially expressed by proliferating cells and are considered as markers for tumorigenesis. We demonstrate that cells lacking RALY expression exhibit changes in the levels of H1FX and ANXA1 mRNAs and proteins in an opposite manner. We also provide evidence for a direct binding of RALY to the U-rich elements present within the 3ÎUTR of both transcripts. Thus, our results identify RALY as a poly-U binding protein and as a regulator of H1FX and ANXA1 in mammalian cells.