RESUMO
In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-UlA and U2 snRNP-U2B". The structure and sequence of SNF, U1A, and U2B" are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B" binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the "RNA-specificity" domain is necessary for SNF's dual function whereas the mixed motif in the U2A'-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Cruzamentos Genéticos , Primers do DNA , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteína Nuclear Pequena U1/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Cromossomo XRESUMO
Stable activation of the Drosophila sex determination gene Sex-lethal in the female embryo is a multistep process. Early in embryogenesis Sex-lethal is regulated at the level of transcription, and then later in embryogenesis Sex-lethal regulation switches to an autoregulatory RNA splicing mechanism. Previous studies have shown that successful activation of Sxl requires both maternally and zygotically provided gene products, many of which are essential for viability and have other, non-sex specific functions. Using a screen for dosage-sensitive modifiers we identified a new maternally expressed gene, l(2)49Db, as a likely participant in Sxl activation. We show that the establishment of the Sxl autoregulatory splicing loop, but not the earlier steps in Sxl activation, is sensitive to the maternal dosage of l(2)49Db. We further demonstrate that l(2)49Db encodes an aspartyl tRNA synthetase. Finally we present evidence that this effect is indirect, by demonstrating that mutations in tryptophanyl tRNA synthetase are also dosage-sensitive maternal modifiers of Sex-lethal. These data suggest that stable activation of Sex-lethal in the embryo may be particularly sensitive to perturbation of the translational machinery.
Assuntos
Aspartato-tRNA Ligase/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes Letais/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Animais , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Drosophila melanogaster/enzimologia , Embrião não Mamífero/metabolismo , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U1/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Processos de Determinação Sexual , Transcrição GênicaRESUMO
Thioredoxin is a highly conserved disulfide reducing protein whose structure and biochemical properties have been extensively studied. Nonetheless, its function in vivo is not well defined. In Drosophila, the maternal-effect gene deadhead encodes a thioredoxin-like protein that is required for initiation of embryonic development. Here we report that deadhead function is dependent on its enzymatic activity: transgenes carrying mutations in thioredoxin's conserved active site failed to rescue the deadhead mutant phenotype. A number of studies have documented that thioredoxin plays a role in DNA synthesis. If thioredoxin is required for DNA synthesis in the fly, then deadhead mutations will suppress mutations that inappropriately synthesize DNA. Contrary to expectation, we find that deadhead does not function as a suppressor in this assay. The observed epistatic relationship between these mutations clearly indicates that deadhead is not essential for DNA metabolism. The possibility of a regulatory role in controlling the initiation of S-phase is discussed.
