Asunto(s)
Drosophila/enzimología , Exorribonucleasas/metabolismo , Animales , Secuencia de Bases , Cromatografía en Gel , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Exorribonucleasas/genética , Exorribonucleasas/aislamiento & purificación , Ácidos Nucleicos/metabolismo , Unión ProteicaRESUMEN
We have identified and characterized a Drosophila orthologue of SKI2, which, in Saccharomyces cerevisiae, is one of the key components in the cytoplasmic 3'-5' decay of mRNA. The Drosophila orthologue (twister, tst), is expressed as two transcripts which differ in the lengths of their 3'-UTRs, with the smaller transcript being particularly abundant in 0-2 h embryos and the larger transcript reaching its highest levels in 6-8 h embryos. TST protein is expressed in two forms which are differentially expressed in adult tissues and throughout development. Differential expression of TST may modulate activity of the mRNA turnover pathway and could have a major impact on the expression of target RNAs.
Asunto(s)
ADN Helicasas , Proteínas de Drosophila , Drosophila/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/genética , Proteínas de Saccharomyces cerevisiae , Animales , Drosophila/embriología , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Etiquetas de Secuencia Expresada , Femenino , Proteínas Fúngicas/genética , Genes de Insecto , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/química , Masculino , Ovario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testículo/metabolismoRESUMEN
In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.