RESUMO
We observed overexpression and increased intra-nuclear accumulation of the PRMT5/WDR77 in breast cancer cell lines relative to immortalized breast epithelial cells. Utilizing mass spectrometry and biochemistry approaches we identified the Zn-finger protein ZNF326, as a novel interaction partner and substrate of the nuclear PRMT5/WDR77 complex. ZNF326 is symmetrically dimethylated at arginine 175 (R175) and this modification is lost in a PRMT5 and WDR77-dependent manner. Loss of PRMT5 or WDR77 in MDA-MB-231 cells leads to defects in alternative splicing, including inclusion of A-T rich exons in target genes, a phenomenon that has previously been observed upon loss of ZNF326. We observed that the alternatively spliced transcripts of a subset of these genes, involved in proliferation and tumor cell migration like REPIN1/AP4, ST3GAL6, TRNAU1AP and PFKM are degraded upon loss of PRMT5. In summary, we have identified a novel mechanism through which the PRMT5/WDR77 complex maintains the balance between splicing and mRNA stability through methylation of ZNF326.
Assuntos
Processamento Alternativo , Proteínas de Transporte/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Células MCF-7 , Ligação Proteica , Proteína-Arginina N-Metiltransferases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Fatores de Transcrição/genéticaRESUMO
Cancer-initiating cells (CIC) undergo asymmetric growth patterns that increase phenotypic diversity and drive selection for chemotherapeutic resistance and tumor relapse. WNT signaling is a hallmark of colon CIC, often caused by APC mutations, which enable activation of ß-catenin and MYC Accumulating evidence indicates that long noncoding RNAs (lncRNA) contribute to the stem-like character of colon cancer cells. In this study, we report enrichment of the lncRNA RBM5-AS1/LUST during sphere formation of colon CIC. Its silencing impaired WNT signaling, whereas its overexpression enforced WNT signaling, cell growth, and survival in serum-free media. RBM5-AS1 has been little characterized previously, and we determined it to be a nuclear-retained transcript that selectively interacted with ß-catenin. Mechanistic investigations showed that silencing or overexpression of RBM5-AS1 caused a respective loss or retention of ß-catenin from TCF4 complexes bound to the WNT target genes SGK1, YAP1, and MYC Our work suggests that RBM5-AS1 activity is critical for the functional enablement of colon cancer stem-like cells. Furthermore, it defines the mechanism of action of RBM5-AS1 in the WNT pathway via physical interactions with ß-catenin, helping organize transcriptional complexes that sustain colon CIC function. Cancer Res; 76(19); 5615-27. ©2016 AACR.
Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias do Colo/patologia , Proteínas de Ligação a DNA/genética , Células-Tronco Neoplásicas/fisiologia , RNA Antissenso/fisiologia , RNA Longo não Codificante/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas Supressoras de Tumor/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Antígeno CD24/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Genes myc , Humanos , Receptores de Hialuronatos/genética , Proteínas Imediatamente Precoces/genética , Camundongos , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição 4 , Fatores de Transcrição/fisiologia , Via de Sinalização Wnt , beta Catenina/fisiologiaRESUMO
The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m(5)C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m(5)C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.
Assuntos
5-Metilcitosina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Elementos Facilitadores Genéticos , Células HEK293 , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Células NIH 3T3 , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
Epigenetic and epitranscriptomic networks have important functions in maintaining the pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However, the mechanisms integrating the actions of these distinct networks are only partially understood. Here we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates the transcription of key pluripotency genes, and modulates N6-methyladenosine (m(6)A) deposition on their transcripts by sequestering the enzyme m(6)A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m(6)A RNA levels, and enhances m(6)A modification of the Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3 associated, and is enriched at promoters of m(6)A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m(6)A RNA modification to ensure ESC identity.