RESUMO
RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.
RESUMO
The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3'-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4-/- (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3'-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.
Assuntos
Processamento Alternativo/genética , Proteína Semelhante a ELAV 4/genética , Neocórtex/metabolismo , Poliadenilação/genética , Animais , Proteína Semelhante a ELAV 4/metabolismo , Éxons/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The neuronal RNA-binding protein (RBP) HuD plays an important role in brain development, synaptic plasticity and neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD). Bioinformatics analysis of the human SOD1 mRNA 3' untranslated region (3'UTR) demonstrated the presence of HuD binding adenine-uridine (AU)-rich instability-conferring elements (AREs). Using differentiated SH-SY5Y cells along with brain tissues from sporadic amyotrophic lateral sclerosis (sALS) patients, we assessed HuD-dependent regulation of SOD1 mRNA. In vitro binding and mRNA decay assays demonstrate that HuD specifically binds to SOD1 ARE motifs promoting mRNA stabilization. In SH-SY5Y cells, overexpression of full-length HuD increased SOD1 mRNA and protein levels while a dominant negative form of the RBP downregulated its expression. HuD regulation of SOD1 mRNA was also found to be oxidative stress (OS)-dependent, as shown by the increased HuD binding and upregulation of this mRNA after H2O2 exposure. This treatment also induced a shift in alternative polyadenylation (APA) site usage in SOD1 3'UTR, increasing the levels of a long variant bearing HuD binding sites. The requirement of HuD for SOD1 upregulation during oxidative damage was validated using a specific siRNA that downregulated HuD protein levels to 36% and prevented upregulation of SOD1 and 91 additional genes. In the motor cortex from sALS patients, we found increases in SOD1 and HuD mRNAs and proteins, accompanied by greater HuD binding to this mRNA as confirmed by RNA-immunoprecipitation (RIP) assays. Altogether, our results suggest a role of HuD in the post-transcriptional regulation of SOD1 expression during ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína Semelhante a ELAV 4/genética , Regulação da Expressão Gênica/genética , Córtex Motor/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/genética , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/metabolismo , Linhagem Celular Tumoral , Proteína Semelhante a ELAV 4/metabolismo , Humanos , RNA Mensageiro/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
The neuronal RNA-binding protein HuD is involved in synaptic plasticity and the molecular mechanisms of learning and memory. Previously, we have shown that HuD is upregulated after both spatial and addiction-associated forms of learning, such as conditioned place preference. However, what role HuD plays in non-drug dependent learning and memory is not fully understood. In order to elucidate the role that HuD plays in non-drug appetitive behavior, we assessed mice over-expressing HuD (HuDOE) throughout the forebrain on the acquisition of an instrumental response for a non-sucrose food reward utilizing a touch-screen paradigm. Next, we examined whether HuD level would alter the extinction or reward-induced reinstatement of responding. We found that HuDOE acquired and extinguished the instrumental response at rates similar to control littermates with no significant alterations in secondary measures of motor behavior or motivation. However, HuDOE reinstated their responding for food reward at rates significantly higher than control animals after a brief presentation of reward. These results suggest that HuD positively regulates the reinstatement of natural reward seeking and supports the role of HuD in forms of learning and memory associated with seeking of appetitive rewards.
Assuntos
Condicionamento Operante/fisiologia , Proteína Semelhante a ELAV 4/biossíntese , Extinção Psicológica/fisiologia , Proteínas de Ligação a RNA/biossíntese , Recompensa , Animais , Proteína Semelhante a ELAV 4/genética , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Prosencéfalo/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.
Assuntos
Regiões 3' não Traduzidas , Axônios/metabolismo , Proteína Semelhante a ELAV 4/metabolismo , Proteína GAP-43/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Animais , Axônios/ultraestrutura , Sequência de Bases , Ligação Competitiva , Proteína Semelhante a ELAV 4/genética , Proteína GAP-43/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Neuropeptídeos/genética , Cultura Primária de Células , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Transdução de SinaisRESUMO
This study investigates the possible mechanism(s) underlying glutathione (GSH) deficiency in the mouse spinal cord during the course of myelin oligodendrocyte glycoprotein35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis. Using the classical enzymatic recycling method and a newly developed immunodot assay, we first demonstrated that total GSH levels (i.e. free GSH plus all its adducts) are reduced in EAE, suggesting an impaired synthesis. The decline in the levels of this essential antioxidant tripeptide in EAE coincides temporally and in magnitude with a reduction in the amount of γ-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis. Other enzymes involved in GSH biosynthesis, whose genes also contain antioxidant-response elements, including glutathione synthetase, cystine/glutamate antiporter, and γ-glutamyl transpeptidase (γ-GT) are diminished in EAE as well. Low levels of γ-glutamylcysteine ligase, glutathione synthetase, and γ-GT are the consequence of reduced mRNA expression, which correlates with diminished expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in both the cytosol and nucleus. Interestingly, the low Nrf2 expression does not seem to be caused by increased degradation via Kelch-like ECH-associated protein 1-dependent or Kelch-like ECH-associated protein 1-independent mechanisms (such as glycogen synthetase kinase-3ß activation), or by reduced levels of Nrf2 mRNA. This suggests that translation of this important transcription factor and/or other still unidentified post-translational processes are altered in EAE. These novel findings are central toward understanding how critical antioxidant and protective responses are lost in inflammatory demyelinating disorders.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Glutationa/deficiência , Fator 2 Relacionado a NF-E2/biossíntese , RNA Mensageiro/biossíntese , Animais , Encefalomielite Autoimune Experimental/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
The single-stranded nucleic acid-binding protein KHSRP (KH-type splicing regulatory protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism, and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer.
Assuntos
Diferenciação Celular , Suscetibilidade a Doenças , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Imunomodulação , Metabolismo dos Lipídeos , Desenvolvimento Muscular , Músculos/fisiologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Transativadores/química , Transativadores/genética , Viroses/imunologia , Viroses/metabolismo , Viroses/virologiaRESUMO
The mammalian embryonic lethal abnormal vision (ELAV)-like protein HuD is a neuronal RNA-binding protein implicated in neuronal development, plasticity, and diseases. Although HuD has long been associated with neuronal development, the functions of HuD in neural stem cell differentiation and the underlying mechanisms have gone largely unexplored. Here we show that HuD promotes neuronal differentiation of neural stem/progenitor cells (NSCs) in the adult subventricular zone by stabilizing the mRNA of special adenine-thymine (AT)-rich DNA-binding protein 1 (SATB1), a critical transcriptional regulator in neurodevelopment. We find that SATB1 deficiency impairs the neuronal differentiation of NSCs, whereas SATB1 overexpression rescues the neuronal differentiation phenotypes resulting from HuD deficiency. Interestingly, we also discover that SATB1 is a transcriptional activator of HuD during NSC neuronal differentiation. In addition, we demonstrate that NeuroD1, a neuronal master regulator, is a direct downstream target of SATB1. Therefore, HuD and SATB1 form a positive regulatory loop that enhances NeuroD1 transcription and subsequent neuronal differentiation. Our results here reveal a novel positive feedback network between an RNA-binding protein and a transcription factor that plays critical regulatory roles in neurogenesis.
Assuntos
Proteína Semelhante a ELAV 4/metabolismo , Retroalimentação Fisiológica , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neurogênese , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Western Blotting , Diferenciação Celular , Células Cultivadas , Proteína Semelhante a ELAV 4/genética , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Dados de Sequência Molecular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Ligação Proteica , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated cis-element sequences of the target mRNA to examine directly protein-RNA interactions in cell-free systems. We found that both ZBP1 and HuD bind the zipcode element in rat ß-actin mRNA's 3' UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3' end of a 5'-ACACCC-3' motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the ß-actin zipcode, but they recognize different features of this target sequence.
Assuntos
Regiões 3' não Traduzidas , Actinas/genética , Proteína Semelhante a ELAV 4/metabolismo , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Actinas/metabolismo , Animais , Neurônios/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RatosRESUMO
The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the "capture" of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the "tag" is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dendritos/metabolismo , Proteínas ELAV/metabolismo , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Dendritos/enzimologia , Dendritos/genética , Proteínas ELAV/genética , Proteína Semelhante a ELAV 4 , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Neurônios/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Ratos , Sinapses/enzimologia , Sinapses/genética , Sinapses/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
The primarily neuronal RNA-binding protein HuD is implicated in learning and memory. Here, we report the identification of several HuD target transcripts linked to Alzheimer's disease (AD) pathogenesis. HuD interacted with the 3' UTRs of APP mRNA (encoding amyloid precursor protein) and BACE1 mRNA (encoding ß-site APP-cleaving enzyme 1) and increased the half-lives of these mRNAs. HuD also associated with and stabilized the long noncoding (lnc)RNA BACE1AS, which partly complements BACE1 mRNA and enhances BACE1 expression. Consistent with HuD promoting production of APP and APP-cleaving enzyme, the levels of APP, BACE1, BACE1AS, and Aß were higher in the brain of HuD-overexpressing mice. Importantly, cortex (superior temporal gyrus) from patients with AD displayed significantly higher levels of HuD and, accordingly, elevated APP, BACE1, BACE1AS, and Aß than did cortical tissue from healthy age-matched individuals. We propose that HuD jointly promotes the production of APP and the cleavage of its amyloidogenic fragment, Aß.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas ELAV/metabolismo , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Córtex Cerebral/metabolismo , Proteínas ELAV/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estabilidade de RNA , RNA Longo não Codificante/genéticaRESUMO
DNA methylation, one of the main epigenetic mechanisms to regulate gene expression, appears to be involved in the development of schizophrenia (SZ). In this study, we investigated 7562 DNA methylation markers in blood from 98 SZ patients and 108 healthy controls. A linear regression model including age, gender, race, alcohol, nicotine and cannabis use status, and diagnosis was implemented to identify C-phosphate-G (CpG) sites significantly associated with diagnosis. These CpG sites were further validated using an independent data set. Sixteen CpG sites were identified with hyper- or hypomethylation in patients. A further verification of expression of the corresponding genes identified 7 genes whose expression levels were also significantly altered in patients. While such altered methylation patterns showed no correlation with disorganized symptoms and negative symptoms in patients, 11 CpG sites significantly correlated with reality distortion symptoms. The direction of the correlations indicates that methylation changes possibly play a protective mechanism to lessen delusion and hallucination symptoms in patients. Pathway analyses showed that the most significant biological function of the differentially methylated CpGs is inflammatory response with CD224, LAX1, TXK, PRF1, CD7, MPG, and MPO genes directly involved in activations of T cells, B cells, and natural killer cells or in cytotoxic reaction. Our results suggest that such methylation changes may modulate aspects of the immune response and hence protect against the neurobiological substrate of reality distortion symptoms in SZ patients.
Assuntos
Metilação de DNA , Epigênese Genética , Esquizofrenia/genética , Psicologia do Esquizofrênico , Adulto , Alcoolismo/epidemiologia , Estudos de Casos e Controles , Ilhas de CpG/genética , Feminino , Predisposição Genética para Doença , Humanos , Inflamação/genética , Modelos Lineares , Masculino , Abuso de Maconha/epidemiologia , Esquizofrenia/epidemiologia , Esquizofrenia/metabolismo , Fumar/epidemiologia , Adulto JovemRESUMO
Little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. We provide evidence for a novel mechanism for mTORC1 kinase-dependent translational regulation of the voltage-gated potassium channel Kv1.1 messenger RNA (mRNA). We identified a microRNA, miR-129, that repressed Kv1.1 mRNA translation when mTORC1 was active. When mTORC1 was inactive, we found that the RNA-binding protein, HuD, bound to Kv1.1 mRNA and promoted its translation. Unexpectedly, inhibition of mTORC1 activity did not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA. However, reduced mTORC1 signaling caused the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Hence, mTORC1 activity regulation of mRNA stability and high affinity HuD-target mRNA degradation mediates the bidirectional expression of dendritic Kv1.1 ion channels.
Assuntos
Proteínas ELAV/metabolismo , Canal de Potássio Kv1.1/metabolismo , MicroRNAs/metabolismo , Proteínas/metabolismo , Proteólise , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação , Proteínas ELAV/genética , Proteína Semelhante a ELAV 4 , Ativação Enzimática , Células HEK293 , Humanos , Canal de Potássio Kv1.1/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , MicroRNAs/genética , Complexos Multiproteicos , Plasticidade Neuronal , Neurônios/metabolismo , Biossíntese de Proteínas , Proteínas/genética , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/genética , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR , TransfecçãoRESUMO
Previous work from our laboratory implicated protein carbonylation in the pathophysiology of both MS (multiple sclerosis) and its animal model EAE (experimental autoimmune encephalomyelitis). Subsequent in vitro studies revealed that the accumulation of protein carbonyls, triggered by glutathione deficiency or proteasome inhibition, leads to protein aggregation and neuronal cell death. These findings prompted us to investigate whether their association can be also established in vivo. In the present study, we characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of MOG (myelin-oligodendrocyte glycoprotein)(35-55) peptide-induced EAE in C57BL/6 mice. The results show that protein carbonyls accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. We also show a temporal correlation between protein carbonylation (but not oxidative stress) and apoptosis. Furthermore, carbonyl levels are significantly higher in apoptotic cells than in live cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are present during the course of EAE. The LC3 (microtubule-associated protein light chain 3)-II/LC3-I ratio is significantly reduced in both acute and chronic EAE indicating reduced autophagy and explaining why aggresomes accumulate in this disorder. Taken together, the results of the present study suggest a link between protein oxidation and neuronal/glial cell death in vivo, and also demonstrate impaired proteostasis in this widely used murine model of MS.
Assuntos
Apoptose/fisiologia , Encefalomielite Autoimune Experimental , Carbonilação Proteica/fisiologia , Medula Espinal/patologia , Animais , Autofagia/fisiologia , Calpaína/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Adjuvante de Freund/imunologia , Adjuvante de Freund/toxicidade , Glutationa/metabolismo , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/toxicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de TempoRESUMO
Complex regulation of brain-derived neurotrophic factor (BDNF) governs its intricate functions in brain development and neuronal plasticity. Besides tight transcriptional control from multiple distinct promoters, alternative 3'end processing of the BDNF transcripts generates either a long or a short 3'untranslated region (3'UTR). Previous reports indicate that distinct RNA sequence in the BDNF 3'UTRs differentially regulates BDNF production in the brain to accommodate neuronal activity changes, conceivably through differential interactions with undefined trans-acting factors that regulate stability and translation of these BDNF mRNA isoforms. In this study, we report that the neuronal RNA-binding protein (RBP) HuD interacts with a highly conserved AU-rich element (ARE) specifically located in the BDNF long 3'UTR. Such interaction is necessary and sufficient for selective stabilization of mRNAs that contain the BDNF long 3'UTR in vitro and in vivo. Moreover, in a HuD transgenic mouse model, the BDNF long 3'UTR mRNA is increased in the hippocampal dentate granule cells (DGCs), leading to elevated expression of BDNF protein that is transported and stored in the mossy fiber (MF) terminals. Our results identify HuD as the first trans-acting factor that enhances BDNF expression specifically through the long 3'UTR and a novel mechanism that regulates BDNF protein production in selected neuronal populations by HuD abundance.
Assuntos
Regiões 3' não Traduzidas , Fator Neurotrófico Derivado do Encéfalo/genética , Encéfalo/metabolismo , Proteínas ELAV/metabolismo , Neurônios/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Genes Reporter , Hipocampo/metabolismo , Camundongos , Ligação Proteica , Interferência de RNA , Elementos de RespostaRESUMO
Hu proteins are a family of RNA-binding proteins (RBPs) that are homologs of Drosophila ELAV, a protein required for nervous system development. Three of these proteins (HuB, HuC, and HuD) are primarily expressed in neurons. The fourth member, HuR is ubiquitously expressed in all tissues. At the molecular level, Hu proteins are known to interact with AU-rich instability conferring sequences in the 3' UTR of specific target mRNAs, stabilizing the mRNAs. These proteins are not only the best known mRNA stabilizers but also the earliest markers of the neuronal cell lineage. Among the neuronal Hu proteins, HuD has been shown to accelerate neuronal differentiation and axonal outgrowth in neurons both in culture and in vivo. In addition, HuD and other Hu proteins participate in synaptic plasticity mechanisms in the mature central nervous system and promote regeneration of peripheral nerves. Furthermore, HuD has been implicated in pathological conditions from neurodegenerative disorders such as Parkinson's and Alzheimer's disease to childhood brain tumors. This review will focus on the involvement of HuD in nervous system function and pathology.
Assuntos
Proteínas ELAV/fisiologia , Animais , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Humanos , Fenômenos Fisiológicos do Sistema Nervoso , Neurônios/metabolismo , Neurônios/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Although expression of the mammalian RNA-binding protein HuD was considered to be restricted to neurons, we report that HuD is present in pancreatic ß cells, where its levels are controlled by the insulin receptor pathway. We found that HuD associated with a 22-nucleotide segment of the 5' untranslated region (UTR) of preproinsulin (Ins2) mRNA. Modulating HuD abundance did not alter Ins2 mRNA levels, but HuD overexpression decreased Ins2 mRNA translation and insulin production, and conversely, HuD silencing enhanced Ins2 mRNA translation and insulin production. Following treatment with glucose, HuD rapidly dissociated from Ins2 mRNA and enabled insulin biosynthesis. Importantly, HuD-knockout mice displayed higher insulin levels in pancreatic islets, while HuD-overexpressing mice exhibited lower insulin levels in islets and in plasma. In sum, our results identify HuD as a pivotal regulator of insulin translation in pancreatic ß cells.
Assuntos
Proteínas ELAV/metabolismo , Insulina/genética , Insulina/metabolismo , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Animais , Proteínas ELAV/genética , Proteína Semelhante a ELAV 4 , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Precursores de Proteínas/genéticaRESUMO
The neuronal RNA-binding protein HuD plays a critical role in the post-transcriptional regulation of short-lived mRNAs during the initial establishment and remodelling of neural connections. We have generated transgenic mice overexpressing this protein (HuD-Tg) in adult DGCs (dentate granule cells) and shown that their mossy fibres contain high levels of GAP-43 (growth-associated protein 43) and exhibit distinct morphological and electrophysiological properties. To investigate the basis for these changes and identify other molecular targets of HuD, DGCs from HuD-Tg and control mice were collected by LCM (laser capture microscopy) and RNAs analysed using DNA microarrays. Results show that 216 known mRNAs transcripts and 63 ESTs (expressed sequence tags) are significantly up-regulated in DGCs from these transgenic mice. Analyses of the 3'-UTRs (3'-untranslated regions) of these transcripts revealed an increased number of HuD-binding sites and the presence of several known instability-conferring sequences. Among these, the mRNA for TTR (transthyretin) shows the highest level of up-regulation, as confirmed by qRT-PCR (quantitative reverse transcription-PCR) and ISH (in situ hybridization). GO (gene ontology) analyses of up-regulated transcripts revealed a large over-representation of genes associated with neural development and axogenesis. In correlation with these gene expression changes, we found an increased length of the infrapyramidal mossy fibre bundle in HuD-Tg mice. These results support the notion that HuD stabilizes a number of developmentally regulated mRNAs in DGCs, resulting in increased axonal elongation.
Assuntos
Axônios/fisiologia , Giro Denteado/citologia , Proteínas ELAV/metabolismo , Camundongos Transgênicos , Regiões 3' não Traduzidas , Animais , Proteínas ELAV/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Masculino , Camundongos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
PURPOSE: An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as 'global regulators' of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses>2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. METHODS: Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT-PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). RESULTS: We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. CONCLUSIONS: The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype.
Assuntos
Proteínas ELAV/genética , Cristalino/metabolismo , MicroRNAs/genética , Neurônios/citologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas Repressoras/genética , Envelhecimento/metabolismo , Processamento Alternativo/genética , Animais , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas ELAV/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Carpa Dourada/genética , Cristalino/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/metabolismo , Mitose , Dados de Sequência Molecular , Fibras Nervosas/metabolismo , Neurônios/metabolismo , Especificidade de Órgãos/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/metabolismo , Ativação Transcricional/genéticaRESUMO
HuD is a neuronal ELAV-like RNA-binding protein (RBP) involved in nervous system development, regeneration, and learning and memory. This protein stabilizes mRNAs by binding to AU-rich instability elements (AREs) in their 3' unstranslated regions (3' UTR). To isolate its in vivo targets, messenger ribonucleoprotein (mRNP) complexes containing HuD were first immunoprecipitated from brain extracts and directly bound mRNAs identified by subsequent GST-HuD pull downs and microarray assays. Using the 3' UTR sequences of the most enriched targets and the known sequence restrictions of the HuD ARE-binding site, we discovered three novel recognition motifs. Motifs 2 and 3 are U-rich whereas motif 1 is C-rich. In vitro binding assays indicated that HuD binds motif 3 with the highest affinity, followed by motifs 2 and 1, with less affinity. These motifs were found to be over-represented in brain mRNAs that are upregulated in HuD overexpressor mice, supporting the biological function of these sequences. Gene ontology analyses revealed that HuD targets are enriched in signaling pathways involved in neuronal differentiation and that many of these mRNAs encode other RBPs, translation factors and actin-binding proteins. These findings provide further insights into the post-transcriptional mechanisms by which HuD promotes neural development and synaptic plasticity.