RESUMEN
The mu opioid receptor has a distinct place in the opioid receptor family, since it mediates the actions of most opioids used clinically (e.g., morphine and fentanyl), as well as drugs of abuse (e.g., heroin). The single-copy mu opioid receptor gene, OPRM1, goes through extensive alternative pre-mRNA splicing to generate numerous splice variants that are conserved from rodents to humans. These OPRM1 splice variants can be classified into three structurally distinct types: (1) full-length 7 transmembrane (TM) carboxyl (C)-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Distinct pharmacological functions of these splice variants have been demonstrated by both in vitro and in vivo studies, particularly by using several unique gene-targeted mouse models. These studies provide new insights into our understanding of the complex actions of mu opioids with regard to OPRM1 alternative splicing. This review provides an overview of the studies that used these gene-targeted mouse models for exploring the functional importance of Oprm1 splice variants.
Asunto(s)
Analgésicos Opioides , Receptores Opioides mu , Empalme Alternativo , Analgésicos Opioides/farmacología , Animales , Ratones , Modelos Animales , Morfina/farmacología , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismoRESUMEN
Most opioid analgesics used clinically, including morphine and fentanyl, as well as the recreational drug heroin, act primarily through the mu opioid receptor, a class A Rhodopsin-like G protein-coupled receptor (GPCR). The single-copy mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating multiple splice variants or isoforms via a variety of alternative splicing events. These OPRM1 splice variants can be categorized into three major types based on the receptor structure: (1) full-length 7 transmembrane (TM) C-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating the distinct actions of various mu opioids. More importantly, the OPRM1 variants can be targeted for development of novel opioid analgesics that are potent against multiple types of pain, but devoid of many side-effects associated with traditional opiates. In this review, we provide an overview of OPRM1 alternative splicing and its functional relevance in opioid pharmacology.
Asunto(s)
Empalme Alternativo/genética , Dolor/genética , Precursores del ARN/genética , Receptores Opioides mu/genética , Analgésicos Opioides/química , Analgésicos Opioides/uso terapéutico , Humanos , Morfina/química , Morfina/uso terapéutico , Dolor/tratamiento farmacológico , Dolor/patología , Isoformas de Proteínas/genética , Empalme del ARN/genéticaRESUMEN
Regulation of alternative splicing can be harnessed by antisense-based compounds to control gene expression. Antisense-mediated splicing interference has become a valuable molecular tool to modulate endogenous alternative splicing patterns, to correct cryptic or aberrant splicing, to reduce gene expression by triggering nonsense-mediated mRNA decay, and to activate intronic polyadenylation, both in vitro and in vivo. Here, we describe methods to induce and analyze the modulation of RNA processing, using modified splice-switching antisense oligonucleotides, such as phosphorodiamidate morpholino (PMO).
Asunto(s)
Empalme Alternativo , Degradación de ARNm Mediada por Codón sin Sentido , Oligodesoxirribonucleótidos Antisentido/química , ARN Mensajero/metabolismo , Animales , HumanosRESUMEN
Extensive 3' alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splice variants. However, their behavioral relevance remains unknown. The present study generated 3 mutant mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129). One mouse truncated all C termini downstream of Oprm1 exon 3 (mE3M mice), while the other two selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice). Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7-associated truncation diminished morphine tolerance and reward without altering physical dependence, whereas the exon 4-associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement in morphine tolerance. In cell-based studies, exon 7-associated variants shifted the bias of several mu opioids toward ß-arrestin 2 over G protein activation compared with the exon 4-associated variant, suggesting an interaction of exon 7-associated C-terminal tails with ß-arrestin 2 in morphine-induced desensitization and tolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacological importance of OPRM1 3' alternative splicing.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Receptores Opioides mu/metabolismo , Empalme Alternativo , Animales , Encéfalo/metabolismo , Codón sin Sentido , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Exones , Tránsito Gastrointestinal/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Dependencia de Morfina/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Opioides mu/genéticaRESUMEN
Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction.
Asunto(s)
Dependencia de Heroína/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Intrones , Polimorfismo de Nucleótido Simple , Empalme del ARN , Receptores Opioides mu/genética , Alelos , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos , Dependencia de Heroína/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Desequilibrio de Ligamiento , Masculino , Receptores Opioides mu/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3' splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-of-function mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner.
Asunto(s)
Genes p53 , Metástasis de la Neoplasia/genética , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Empalme Alternativo , Animales , Antígeno CD24/metabolismo , Cadherinas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Peptidil-Prolil Isomerasa F , Ciclofilinas/metabolismo , Transición Epitelial-Mesenquimal/genética , Humanos , Receptores de Hialuranos/metabolismo , Intrones , Lesión Pulmonar/genética , Lesión Pulmonar/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Mitocondrias/metabolismo , Mutación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sitios de Empalme de ARN , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5' splice site. U1 snRNP also plays an additional, nonsplicing global function in 3' end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3' end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer.
RESUMEN
ERG, a member of the ETS transcription factor family, is frequently overexpressed in prostate cancer as a result of its fusion to the androgen-responsive Tmprss2 gene. Different genomic rearrangements and alternative splicing events around the junction region lead to multiple combination of Tmprss2:ERG fusion transcripts that correlate with different tumor aggressiveness, but their specific functions and biological activities are still unclear. The complexity of ERG expression pattern is compounded by the use of alternative promoters, splice sites, polyadenylation sites and translation initiation sites in both the native and fusion contexts. Our systematic characterization of native ERG and Tmprss2:ERG variants reveals that their different oncogenic potential is impacted by the status of the Ets domain and the configuration of the 5' UTR region. In particular, expression and activity of functional ERG and Tmprss2:ERG variants are influenced both by translation initiation signals within the different isoforms and by inhibitory upstream Open Reading Frames (uORF) in their 5' UTRs. Stable expression of ERG and Tmprss2:ERG variants promoted cell migration/invasion, induced a block of proliferation and induced a senescence-like state, suggesting a role for these variants in the prostate tumorigenesis process. In addition to Tmprss2:ERG fusion products, a group of related native ERG isoforms is also highly over-expressed in fusion-carrying prostate cancers, and share the same translation initiation site (in ERG exon 4) with the commonly observed Tmprss2 exon1 joined to ERG exon 4 (T1:E4) fusion-derived variant. Usage of this ATG can be preferentially down-regulated by directed antisense-based compounds, possibly representing the basis of a targeted approach that distinguishes between tumor-associated and normal ERG.
Asunto(s)
Regiones no Traducidas 5' , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Serina Endopeptidasas/genética , Transactivadores/genética , Empalme Alternativo , Animales , Muerte Celular , Línea Celular Tumoral , Variación Genética , Células HeLa , Humanos , Masculino , Ratones , Células 3T3 NIH , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Regulador Transcripcional ERGRESUMEN
Next-generation antisense technologies are re-emerging as viable and powerful approaches to the treatment of several genetic diseases. Similar strategies are also being applied to cancer therapy. Re-programming of the expression of endogenous oncogenic products to replace them with functional antagonists, by interfering with alternative splicing or polyadenylation, provides a promising novel approach to address acquired drug resistance and previously undruggable targets.
RESUMEN
Preclinical and clinical studies demonstrate the feasibility of treating ß-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human ß-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human ß-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human ß-globin through a novel mechanism that links the rate of transcription of the transgenic ß-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34(+) cells isolated from patients affected by ß-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).Our results suggest two major findings. First, we discovered that for the purpose of expressing the ß-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from ß-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.
Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética/métodos , Hemoglobinas/metabolismo , Talasemia beta/terapia , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Animales , Ancirinas/genética , Antígenos CD34/metabolismo , Secuencia de Bases , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Células Precursoras Eritroides/metabolismo , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Hemoglobinas/genética , Humanos , Elementos Aisladores/genética , Lentivirus/genética , Ratones , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Globinas beta/genética , Talasemia beta/sangre , Talasemia beta/genéticaRESUMEN
Signal transducer and activator of transcription 3 (STAT3) plays a central role in the activation of multiple oncogenic pathways. Splicing variant STAT3ß uses an alternative acceptor site within exon 23 that leads to a truncated isoform lacking the C-terminal transactivation domain. Depending on the context, STAT3ß can act as a dominant-negative regulator of transcription and promote apoptosis. We show that modified antisense oligonucleotides targeted to a splicing enhancer that regulates STAT3 exon 23 alternative splicing specifically promote a shift of expression from STAT3α to STAT3ß. Induction of endogenous STAT3ß leads to apoptosis and cell-cycle arrest in cell lines with persistent STAT3 tyrosine phosphorylation compared with total STAT3 knockdown obtained by forced splicing-dependent nonsense-mediated decay (FSD-NMD). Comparison of the molecular effects of splicing redirection to STAT3 knockdown reveals a unique STAT3ß signature, with a down-regulation of specific targets (including lens epithelium-derived growth factor, p300/CBP-associated factor, CyclinC, peroxisomal biogenesis factor 1, and STAT1ß) distinct from canonical STAT3 targets typically associated with total STAT3 knockdown. Furthermore, similar in vivo redirection of STAT3 alternative splicing leads to tumor regression in a xenograft cancer model, demonstrating how pharmacological manipulation of a single key splicing event can manifest powerful antitumorigenic properties and validating endogenous splicing reprogramming as an effective cancer therapeutic approach.
Asunto(s)
Empalme Alternativo/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Animales , Western Blotting , Línea Celular , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , Análisis por Micromatrices , Oligonucleótidos Antisentido/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong antiangiogenic effect both on directly targeted cells and on naive cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.
Asunto(s)
Intrones , Poliadenilación , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Humanos , Neovascularización Fisiológica/fisiología , Poli A/química , Poli A/genética , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , Empalme del ARN , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/fisiología , Ribonucleoproteína Nuclear Pequeña U1/fisiología , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-α/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur d'Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16- or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Empalme Alternativo , Animales , Corteza Cerebral/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Exones , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Empalme del ARN , Elementos Reguladores de la TranscripciónRESUMEN
Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Genes Supresores de Tumor , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Pancreáticas/genética , Secuencia de Bases , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Supervivencia Celular , Cromosomas Humanos Par 9/genética , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Pérdida de Heterocigocidad , Neoplasias Pancreáticas/patología , Biosíntesis de Proteínas/genética , Proteínas RGSRESUMEN
Lentiviral-mediated beta-globin gene transfer successfully treated beta-thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different beta-globin mutations found in patients. Most mutations can be classified as beta(0) or beta(+), based on the amount of beta-globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two-phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human beta-globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooley's anemia patients.
Asunto(s)
Terapia Genética , Talasemia beta/genética , Talasemia beta/terapia , Animales , Células Cultivadas , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Humanos , Lentivirus/genética , Ratones , Mutación , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Transcription factors are important targets for the treatment of a variety of malignancies but are extremely difficult to inhibit, as they are located in the cell's nucleus and act mainly by protein-DNA and protein-protein interactions. The transcriptional regulators Id1 and Id3 are attractive targets for cancer therapy as they are required for tumor invasiveness, metastasis and angiogenesis. We report here the development of an antitumor agent that downregulates Id1 effectively in tumor endothelial cells in vivo. Efficient delivery and substantial reduction of Id1 protein levels in the tumor endothelium were effected by fusing an antisense molecule to a peptide known to home specifically to tumor neovessels. In two different tumor models, systemic delivery of this drug led to enhanced hemorrhage, hypoxia and inhibition of primary tumor growth and metastasis, similar to what is observed in Id1 knockout mice. Combination with the Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin yielded virtually complete growth suppression of aggressive breast tumors.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Marcación de Gen/métodos , Proteínas de Neoplasias/genética , Oligonucleótidos Antisentido/genética , Péptidos/genética , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Ratones , Oligonucleótidos Antisentido/administración & dosificación , Péptidos/administración & dosificaciónRESUMEN
Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GR alpha and GR beta isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GR beta, but not GR alpha. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GR beta expression and contributing to glucocorticoid resistance in PC cells.
Asunto(s)
Bombesina/metabolismo , Neurotransmisores/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Receptores de Glucocorticoides/metabolismo , Bombesina/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neurotransmisores/genética , Proteínas Nucleares/genética , Neoplasias de la Próstata , Isoformas de Proteínas/genética , Precursores del ARN/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Receptores de Glucocorticoides/genética , Factores de Empalme Serina-ArgininaRESUMEN
The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3' splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on splicing be evaluated in the context of the relevant haplotype.
Asunto(s)
Acil-CoA Deshidrogenasa/genética , Elementos de Facilitación Genéticos/genética , Exones/genética , Errores Innatos del Metabolismo Lipídico/inmunología , Polimorfismo de Nucleótido Simple , Empalme del ARN/genética , Elementos Silenciadores Transcripcionales/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Cartilla de ADN/genética , Femenino , Genes BRCA1/fisiología , Humanos , Inmunidad , Lactante , Recién Nacido , Errores Innatos del Metabolismo Lipídico/genética , Datos de Secuencia Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/inmunología , Mutación Missense/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Estabilidad del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN , Homología de Secuencia de Ácido Nucleico , Proteína 2 para la Supervivencia de la Neurona Motora , Transcripción GenéticaRESUMEN
Spinal muscular atrophy is a neurodegenerative disorder caused by the deletion or mutation of the survival-of-motor-neuron gene, SMN1. An SMN1 paralog, SMN2, differs by a C-->T transition in exon 7 that causes substantial skipping of this exon, such that SMN2 expresses only low levels of functional protein. A better understanding of SMN splicing mechanisms should facilitate the development of drugs that increase survival motor neuron (SMN) protein levels by improving SMN2 exon 7 inclusion. In addition, exonic mutations that cause defective splicing give rise to many genetic diseases, and the SMN1/2 system is a useful paradigm for understanding exon-identity determinants and alternative-splicing mechanisms. Skipping of SMN2 exon 7 was previously attributed either to the loss of an SF2/ASF-dependent exonic splicing enhancer or to the creation of an hnRNP A/B-dependent exonic splicing silencer, as a result of the C-->T transition. We report the extensive testing of the enhancer-loss and silencer-gain models by mutagenesis, RNA interference, overexpression, RNA splicing, and RNA-protein interaction experiments. Our results support the enhancer-loss model but also demonstrate that hnRNP A/B proteins antagonize SF2/ASF-dependent ESE activity and promote exon 7 skipping by a mechanism that is independent of the C-->T transition and is, therefore, common to both SMN1 and SMN2. Our findings explain the basis of defective SMN2 splicing, illustrate the fine balance between positive and negative determinants of exon identity and alternative splicing, and underscore the importance of antagonistic splicing factors and exonic elements in a disease context.