RESUMO
Tumor necrosis factor-α (TNF-α) is the most potent proinflammatory cytokine in mammals. The degradation of TNF-α mRNA is critical for restricting TNF-α synthesis and involves a constitutive decay element (CDE) in the 3' UTR of the mRNA. Here, we demonstrate that the CDE folds into an RNA stem-loop motif that is specifically recognized by Roquin and Roquin2. Binding of Roquin initiates degradation of TNF-α mRNA and limits TNF-α production in macrophages. Roquin proteins promote mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. CDE sequences are highly conserved and are found in more than 50 vertebrate mRNAs, many of which encode regulators of development and inflammation. In macrophages, CDE-containing mRNAs were identified as the primary targets of Roquin on a transcriptome-wide scale. Thus, Roquin proteins act broadly as mediators of mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs.
Assuntos
Macrófagos/metabolismo , Estabilidade de RNA , Proteínas Repressoras/metabolismo , Fator de Necrose Tumoral alfa/genética , Ubiquitina-Proteína Ligases/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular , Humanos , Inflamação/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , RNA Mensageiro/química , Alinhamento de SequênciaRESUMO
Acetylation of histones and transcription-related factors is known to exert epigenetic and transcriptional control of gene expression. Here we report that histone acetyltransferases (HATs) and histone deacetylases (HDACs) also regulate gene expression at the posttranscriptional level by controlling poly(A) RNA stability. Inhibition of HDAC1 and HDAC2 induces massive and widespread degradation of normally stable poly(A) RNA in mammalian and Drosophila cells. Acetylation-induced RNA decay depends on the HATs p300 and CBP, which acetylate the exoribonuclease CAF1a, a catalytic subunit of the CCR4-CAF1-NOT deadenlyase complex and thereby contribute to accelerating poly(A) RNA degradation. Taking adipocyte differentiation as a model, we observe global stabilization of poly(A) RNA during differentiation, concomitant with loss of CBP/p300 expression. Our study uncovers reversible acetylation as a fundamental switch by which HATs and HDACs control the overall turnover of poly(A) RNA.
Assuntos
Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Poli A/genética , RNA Mensageiro/genética , Fatores de Transcrição de p300-CBP/genética , Células 3T3-L1 , Acetilação , Sequência de Aminoácidos , Animais , Diferenciação Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Camundongos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Poli A/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
SIRT7 is an NAD+-dependent protein deacetylase that regulates cell growth and proliferation. Previous studies have shown that SIRT7 is required for RNA polymerase I (Pol I) transcription and pre-rRNA processing. Here, we took a proteomic approach to identify novel molecular targets and characterize the role of SIRT7 in non-nucleolar processes. We show that SIRT7 interacts with numerous proteins involved in transcriptional regulation and RNA metabolism, the majority of interactions requiring ongoing transcription. In addition to its role in Pol I transcription, we found that SIRT7 also regulates transcription of snoRNAs and mRNAs. Mechanistically, SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex and deacetylates CDK9, a subunit of the elongation factor P-TEFb, which activates transcription by phosphorylating serine 2 within the C-terminal domain (CTD) of Pol II. SIRT7 counteracts GCN5-directed acetylation of lysine 48 within the catalytic domain of CDK9, deacetylation promoting CTD phosphorylation and transcription elongation.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , RNA Polimerase II/metabolismo , Sirtuínas/metabolismo , Ativação Transcricional , Linhagem Celular , Humanos , Fator B de Elongação Transcricional Positiva/metabolismo , RNA/metabolismo , RNA Nucleolar Pequeno/biossíntese , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Sirtuínas/químicaRESUMO
Activation of γ-aminobutyric A receptors (GABA(A)Rs) in the subependymal zone (SEZ) induces hyperpolarization and osmotic swelling in precursors, thereby promoting surface expression of the epidermal growth factor receptor (EGFR) and cell cycle entry. However, the mechanisms underlying the GABAergic modulation of cell swelling are unclear. Here, we show that GABA(A)Rs colocalize with the water channel aquaporin (AQP) 4 in prominin-1 immunopositive (P(+)) precursors in the postnatal SEZ, which include neural stem cells. GABA(A)R signaling promotes AQP4 expression by decreasing serine phosphorylation associated with the water channel. The modulation of AQP4 expression by GABA(A)R signaling is key to its effect on cell swelling and EGFR expression. In addition, GABA(A)R function also affects the ability of neural precursors to swell in response to an osmotic challenge in vitro and in vivo. Thus, the regulation of AQP4 by GABA(A)Rs is involved in controlling activation of neural stem cells and water exchange dynamics in the SEZ.
Assuntos
Aquaporina 4/fisiologia , Epêndima/metabolismo , Regulação da Expressão Gênica , Ventrículos Laterais/metabolismo , Receptores de GABA-A/metabolismo , Água/metabolismo , Animais , Western Blotting , Tamanho Celular , Células Cultivadas , Epêndima/citologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Imunofluorescência , Imunoprecipitação , Ventrículos Laterais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pressão Osmótica , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de GABA-A/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
BACKGROUND: Cytoplasmic polyadenylation element-binding protein 4 (CPEB4) is known to associate with cytoplasmic polyadenylation elements (CPEs) located in the 3' untranslated region (UTR) of specific mRNAs and assemble an activator complex promoting the translation of target mRNAs through cytoplasmic polyadenylation. RESULTS: Here, we find that CPEB4 is part of an alternative repressor complex that mediates mRNA degradation by associating with the evolutionarily conserved CCR4-NOT deadenylase complex. We identify human CPEB4 as an RNA-binding protein (RBP) with enhanced association to poly(A) RNA upon inhibition of class I histone deacetylases (HDACs), a condition known to cause widespread degradation of poly(A)-containing mRNA. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis using endogenously tagged CPEB4 in HeLa cells reveals that CPEB4 preferentially binds to the 3'UTR of immediate early gene mRNAs, at G-containing variants of the canonical U- and A-rich CPE located in close proximity to poly(A) sites. By transcriptome-wide mRNA decay measurements, we find that the strength of CPEB4 binding correlates with short mRNA half-lives and that loss of CPEB4 expression leads to the stabilization of immediate early gene mRNAs. Akin to CPEB4, we demonstrate that CPEB1 and CPEB2 also confer mRNA instability by recruitment of the CCR4-NOT complex. CONCLUSIONS: While CPEB4 was previously known for its ability to stimulate cytoplasmic polyadenylation, our findings establish an additional function for CPEB4 as the RNA adaptor of a repressor complex that enhances the degradation of short-lived immediate early gene mRNAs.
Assuntos
Genes Precoces , Estabilidade de RNA , Regiões 3' não Traduzidas , Células HeLa , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.