RESUMEN
The patterns of transcription and translation of the ribosomal protein L32 (Rpl32) mRNA differ greatly in adult testis and somatic tissues. Northern blots reveal that the levels of Rpl32 mRNA are four- to five-fold higher in prepubertal and adult testes, and purified pachytene spermatocytes and round spermatids than in a variety of nongrowing adult somatic tissues. 5' RACE demonstrates that transcription in 8-day prepubertal testis, which lacks meiotic and haploid cells, strongly prefers the same start site in the 5' terminal oligopyrimidine tract (5' TOP) that is used is somatic cells. The 5' TOP is a cis element that inhibits translation of many mRNAs in nongrowing somatic cells. Although the sizes of deadenylated Rpl32 mRNAs are indistinguishable in somatic and spermatogenic cells, transcription initiates at 11 sites over a 31-nt segment in adult testis and approximately 62% of Rpl32 mRNAs lack a 5' TOP. In agreement with previous studies, low levels of cycloheximide increase the proportions and sizes of polysomes in absorbance profiles, and increase the proportions and sizes of polysomes translating four 5' TOP mRNA species including the Rpl32 mRNA in 8-day seminiferous tubules. In contrast, cycloheximide has little or no effect on the absorbance profiles and distribution of Rpl32 mRNA and 5' TOP mRNAs in adult seminiferous tubules. The failure of cycloheximide to increase the size of polysomes in adult seminiferous tubules implies a block in the pathway by which ribosomes are recruited onto translationally active mRNAs.
Asunto(s)
ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Espermatocitos/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Codón de Terminación/genética , Cicloheximida/farmacología , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Polirribosomas/efectos de los fármacos , Polirribosomas/genética , Polirribosomas/metabolismo , Biosíntesis de Proteínas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Secuencia de Oligopirimidina en la Región 5' Terminal del ARN/genética , ARN Mensajero/genética , Espermátides/citología , Espermátides/metabolismo , Espermatocitos/citología , Testículo/citología , Transcripción Genética/genéticaRESUMEN
Megakaryocytes, the platelet precursors, are induced to differentiate in response to Mpl ligand. Here we report that stability of the megakaryocyte-specific platelet factor 4 (PF4) mRNA is substantially augmented in the presence of Mpl ligand. This stabilization requires protein synthesis, but the 3'-untranslated region of PF4 mRNA is not sufficient for granting the effect. This cytokine also significantly or mildly stabilizes Mpl receptor or GAPDH mRNAs, respectively, in contrast to a previously reported lack of effect on P2Y(1) receptor mRNA. Our study is the first to suggest that Mpl ligand-induced lineage specification is also determined by message stabilization.
Asunto(s)
Megacariocitos/citología , Factor Plaquetario 4/genética , Estabilidad del ARN , ARN Mensajero/efectos de los fármacos , Trombopoyetina/farmacología , Región de Flanqueo 3' , Regiones no Traducidas 3' , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Gliceraldehído-3-Fosfato Deshidrogenasas/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Megacariocitos/efectos de los fármacos , Ratones , Proteínas de Neoplasias/genética , Factor Plaquetario 4/efectos de los fármacos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Receptores de Citocinas/genética , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Receptores de TrombopoyetinaRESUMEN
The profile of expression of the A3 adenosine receptor (A3AR) and its importance during embryo development were explored. To this end, different ages of mouse embryos (8.5 days and older) were subjected to in situ hybridization with an A3AR riboprobe. No expression was found in any embryonic tissue except for the aorta and heart of 15.5-day embryos. To investigate further the role of the A3AR gene in development, we overexpressed this gene in A3AR knockout and wild-type mice by using the SM22 alpha promoter. This promoter is active in smooth, cardiac, and skeletal muscle lineages during early embryogenesis (at 8.5 days or earlier), becoming restricted to vascular and visceral smooth muscle cells in late fetal development and adult mice. We observed that moderate copy number incorporation (four copies) of the A3AR gene driven by the SM22 alpha promoter is sufficient to induce lethality at an early stage of embryo development. Remains of 8.5-day transgenic embryos were collected, including fragmented DNA. Hence, we speculate that A3AR homeostasis is critical for embryo viability and proper development. This finding is intriguing in view of the reported effects of sustained activation of the A3AR on induction of DNA fragmentation and apoptosis in cultured myocytes and other cell types.