RESUMO
We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5' splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Íntrons , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Splicing de RNA , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo , Animais , Sequência de Bases , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Purinas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A minor class of pre-mRNA introns whose excision requires a spliceosome containing U11, U12, U4atac/U6atac, and U5 snRNPs has been identified in plants, insects, and vertebrates. We have characterized single loci that specify the U6atac and U12 snRNAs of Drosophila melanogaster. P element-mediated disruptions of the U6atac and U12 genes cause lethality during the third instar larval and embryonic stages, respectively, and are rescued by U6atac and U12 transgenes. The P element disruption of U6atac results in excision defects of U12-type introns from several transcripts including an alternative U12-dependent spliced isoform of prospero, a homeodomain protein required for CNS development. Thus, we demonstrate the requirement for the U12 spliceosome in the development of a metazoan organism.