RESUMO
Nanos protein promotes abdominal structures in Drosophila embryos by repressing the translation of maternal hunchback mRNA in the posterior. To study the mechanism of nanos-mediated translational repression, we first examined the mechanism by which maternal hunchback mRNA is translationally activated. In the absence of nanos activity, the poly(A) tail of hunchback mRNA is elongated concomitant with its translation, suggesting that cytoplasmic polyadenylation directs activation. However, in the presence of nanos the length of the hunchback mRNA poly(A) tail is reduced. To determine if nanos activity represses translation by altering the polyadenylation state of hunchback mRNA, we injected various in vitro transcribed RNAs into Drosophila embryos and determined changes in polyadenylation. Nanos activity reduced the polyadenylation status of injected hunchback RNAs by accelerating their deadenylation. Pumilio activity, which is necessary to repress the translation of hunchback, is also needed to alter polyadenylation. An examination of translation indicates a strong correlation between poly(A) shortening and suppression of translation. These data indicate that nanos and pumilio determine posterior morphology by promoting the deadenylation of maternal hunchback mRNA, thereby repressing its translation.
Assuntos
Padronização Corporal/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/embriologia , Proteínas de Insetos/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição/genética , Animais , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Modelos Genéticos , Biossíntese de Proteínas/fisiologia , Transativadores/genéticaRESUMO
Translational recruitment of maternal mRNAs is an essential process in early metazoan development. To identify genes required for this regulatory pathway, we have examined a collection of Drosophila female-sterile mutants for defects in translation of maternal mRNAs. This strategy has revealed that maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of bicoid and Toll mRNAs. Cortex embryos contain a bicoid mRNA indistinguishable in amount, localization, and structure from that in wild-type embryos. However, the bicoid mRNA in cortex embryos contains a shorter than normal polyadenosine (poly(A)) tail. Injection of polyadenylated bicoid mRNA into cortex embryos allows translation demonstrating that insufficient polyadenylation prevents endogenous bicoid mRNA translation. In contrast nanos mRNA, which is activated by a poly(A)-independent mechanism, is translated in cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. Cortex embryos are fertilized, but arrest at the onset of embryogenesis. Characterization of grauzone mutations indicates that the phenotype of these embryos is similar to cortex. These results identify a fundamental pathway that serves a vital role in the initiation of development.
Assuntos
Drosophila/fisiologia , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Primers do DNA , Drosophila/embriologia , Drosophila/genética , Embrião não Mamífero/fisiologia , Feminino , Genes de Insetos , Infertilidade Feminina , Dados de Sequência Molecular , Mutagênese , Oogênese , Iniciação Traducional da Cadeia Peptídica , Reação em Cadeia da Polimerase , Temperatura , Fatores de TempoRESUMO
Pattern formation in Drosophila depends initially on the translational activation of maternal messenger RNAs (mRNAs) whose protein products determine cell fate. Three mRNAs that dictate anterior, dorsoventral, and terminal specification--bicoid, Toll, and torso, respectively--showed increases in polyadenylate [poly(A)] tail length concomitant with translation. In contrast, posteriorly localized nanos mRNA, although also translationally activated, was not regulated by poly(A) status. These results implicate at least two mechanisms of mRNA activation in flies. Studies with bicoid mRNA showed that cytoplasmic polyadenylation is necessary for translation, establishing this pathway as essential for embryogenesis. Combined, these experiments identify a regulatory pathway that can coordinate initiation of maternal pattern formation systems in Drosophila.
