RESUMEN
Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Oligospermia/complicaciones , Motilidad Espermática , Espermatozoides/anomalías , Animales , Células COS , Factor 1 de Ensamblaje de la Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Femenino , Fibroblastos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Oligospermia/etiología , Oligospermia/genética , Fenotipo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/fisiología , Túbulos Seminíferos/metabolismo , Espermatogénesis/genética , Trasplante de Células MadreRESUMEN
The stability of mRNA influences the abundance of cellular transcripts and proteins. Deadenylases play critical roles in mRNA turnover and thus are important for the regulation of various biological events. Here, we report the identification and characterization of CCR4b/CNOT6L, which is homologous to yeast CCR4 mRNA deadenylase. CCR4b is localized mainly in the cytoplasm and displays deadenylase activity both in vitro and in vivo. CCR4b forms a multisubunit complex similar to the yeast CCR4-NOT complex. Suppression of CCR4b by RNA interference results in growth retardation of NIH 3T3 cells accompanied by elevation of both p27(Kip1) mRNA and p27(Kip1) protein. Reintroduction of wild-type CCR4b, but not mutant CCR4b lacking deadenylase activity, restores the growth of CCR4b-depleted NIH 3T3 cells. The data suggest that CCR4b regulates cell growth in a manner dependent on its deadenylase activity. We also show that p27(Kip1) mRNA is stabilized and its poly(A) tail is preserved in CCR4b-depleted cells. Our findings provide evidence that CCR4b deadenylase is a constituent of the mammalian CCR4-NOT complex and regulates the turnover rate of specific target mRNAs. Thus, CCR4b may be involved in various cellular events that include cell proliferation.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Ribonucleasas/deficiencia , Animales , Ciclo Celular , Línea Celular , Proliferación Celular , Clonación Molecular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , ADN Complementario , Exorribonucleasas/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Células 3T3 NIH , Poli A/metabolismo , Unión Proteica , Transporte de Proteínas , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/química , Fracciones Subcelulares/metabolismo , Distribución TisularRESUMEN
Tob protein, when overexpressed, suppresses growth of NIH3T3 cells, presumably by regulating expression of various growth-related genes. However, the molecular mechanisms underlying Tob-mediated regulation of gene expression have been obscure. To address this issue we established stable Tob-expressing cell lines and used a proteomics approach to identify Tob-interacting proteins. We found that Tob associates with the CCR4-NOT complex. The carboxyl-terminal half of Tob interacted with Cnot1, a core protein of the CCR4-NOT complex. We further showed that the deadenylase activity associated with the complex was suppressed in vitro by Tob. These results suggest that the antiproliferative activity of Tob is shown post-transcriptionally by controlling the stability of the target mRNAs in addition to its involvement in transcriptional regulation, reported previously.
Asunto(s)
Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Estabilidad del ARN , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Células 3T3 NIH , Biosíntesis de Proteínas/genética , Proteómica , Proteínas Supresoras de Tumor/genéticaRESUMEN
tob is a member of antiproliferative family genes. Mice lacking tob are prone to spontaneous formation of tumors. The occurrence rate of diethylnitrosamine-induced liver tumors is higher in tob(-/-) mice than in wild-type mice. tob(-/-)p53(-/-) mice show accelerated tumor formation in comparison with single null mice. Expression of cyclin D1 mRNA is increased in the absence of Tob and is reduced by Tob. Tob acts as a transcriptional corepressor and suppresses the cyclin D1 promoter activity through an interaction with histone deacetylase. Levels of tob mRNA are often decreased in human cancers, implicating tob in cancer development.