RESUMEN
CUG-binding protein, ELAV-like Family Member 1 (CELF1) plays an important role during the development of different tissues, such as striated muscle and brain tissue. CELF1 is an RNA-binding protein that regulates RNA metabolism processes, e.g., alternative splicing, and antagonizes other RNA-binding proteins, such as Muscleblind-like proteins (MBNLs). Abnormal activity of both classes of proteins plays a crucial role in the pathogenesis of myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults. In this work, we show that alternative splicing of exons forming both the 5' and 3' untranslated regions (UTRs) of CELF1 mRNA is efficiently regulated during development and tissue differentiation and is disrupted in skeletal muscles in the context of DM1. Alternative splicing of the CELF1 5'UTR leads to translation of two potential protein isoforms that differ in the lengths of their N-terminal domains. We also show that the MBNL and CELF proteins regulate the distribution of mRNA splicing isoforms with different 5'UTRs and 3'UTRs and affect the CELF1 expression by changing its sensitivity to specific microRNAs or RNA-binding proteins. Together, our findings show the existence of different mechanisms of regulation of CELF1 expression through the distribution of various 5' and 3' UTR isoforms within CELF1 mRNA.
Asunto(s)
Regiones no Traducidas 3' , Regiones no Traducidas 5' , Empalme Alternativo , Proteínas CELF1/biosíntesis , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , ARN Mensajero/metabolismo , Adulto , Anciano , Animales , Sitios de Unión , Proteínas CELF1/genética , Células COS , Chlorocebus aethiops , Exones , Femenino , Células HeLa , Humanos , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Distrofias Musculares/genética , Biosíntesis de Proteínas , Isoformas de Proteínas , ARN Mensajero/genética , Adulto JovenRESUMEN
The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma.
Asunto(s)
Proteínas CELF1/genética , Hepatoblastoma/genética , Neoplasias Hepáticas/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas/genética , Receptores Citoplasmáticos y Nucleares/genética , Animales , Proteínas CELF1/biosíntesis , Diferenciación Celular/genética , Línea Celular Tumoral , Dietilnitrosamina/toxicidad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hepatoblastoma/inducido químicamente , Hepatoblastoma/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Pediatría , Receptores Citoplasmáticos y Nucleares/biosíntesisRESUMEN
RNA binding protein is identified as an important mediator of aberrant alternative splicing in muscle atrophy. The altered splicing of calcium channels, such as ryanodine receptors (RyRs), plays an important role in impaired excitation-contraction (E-C) coupling in muscle atrophy; however, the regulatory mechanisms of ryanodine receptor 1 (RyR1) alternative splicing leading to skeletal muscle atrophy remains to be investigated. In this study we demonstrated that CUG binding protein 1 (CUG-BP1) was up-regulated and the alternative splicing of RyR1 ASI (exon70) was aberrant during the process of neurogenic muscle atrophy both in human patients and mouse models. The gain and loss of function experiments in vivo demonstrated that altered splicing pattern of RyR1 ASI was directly mediated by an up-regulated CUG-BP1 function. Furthermore, we found that CUG-BP1 affected the calcium release activity in single myofibers and the extent of atrophy was significantly reduced upon gene silencing of CUG-BP1 in atrophic muscle. These findings improve our understanding of calcium signaling related biological function of CUG-BP1 in muscle atrophy. Thus, we provide an intriguing perspective of involvement of mis-regulated RyR1 splicing in muscular disease.