Post-transcriptional Regulation of nanos mRNA During Early Drosophila Development

Abstract

Translational control is essential throughout early development of the fruit fly <italic>Drosophila melanogaster</italic> to ensure precise spatial and temporal regulation of gene expression. For example, translation of maternal <italic>nanos (nos)</italic> mRNA must be restricted to the posterior of the embryo to ensure proper patterning of the anterior-posterior body axis. During oogenesis, a subset of <italic>nos </italic>mRNA becomes localized to the posterior of the oocyte, where <italic> nos</italic> translation is activated. This localization is maintained into embryogenesis, as local production of Nos protein is required for abdominal development. The vast majority of <italic>nos </italic>mRNA remains unlocalized and translationally repressed, however. It is essential for this unlocalized mRNA to remain repressed in both the oocyte and early embryo, as the presence of Nos protein at the anterior of the embryo interferes with normal development of head and thorax structures.

Repression in the bulk cytoplasm is mediated by a translational control element (TCE) in the nos 3' untranslated region. The TCE is a structural motif consisting of two major stem loops (II and III) that are bound by the repressor proteins Smaug (Smg) in the embryo and Glorund (Glo) in the ovary. We have shown that <italic>nos </italic>translation in the ovary occurs by two mechanisms, one that acts at the initiation phase and one that acts post-initiation, as repressed <italic>nos</italic> mRNA remains associated with polyribosomes in late stage oocytes. Our results suggest that both of these mechanisms are mediated by Glo and its interacting partners. Although the post-initiation block is maintained into embryogenesis, subsequent translational initiation in the embryo is then repressed at initiation by Smg and its interacting partners.

Although Glo was initially identified by its specific interaction with TCE stem loop III, the pleotropic nature of the mutant phenotype suggests that Glo functions in various cellular processes, including mRNA localization and nurse cell chromatin dispersion. In order to more fully understand what dictates the distinct activities of Glo, we performed mutagenesis of conserved residues within the quasi RNA recognition motifs (qRRMs) of Glo. Despite a lack of conservation in overall sequence between Glo and its human homologs, we have demonstrated that the qRRM domains share G-quartet RNA binding activity with its homologs. However, TCE stem loop III binding may be mediated by unique residues within the qRRM.

While there is an abundance of data demonstrating that the TCE modulates <italic>nos </italic>translation throughout early development by its interactions with Glo and Smg, there is evidence that additional trans-acting factors are likely to be involved. These factors, through their binding to the 3'UTR, may contribute to the post-initiation phase of nos regulation. Through the use of the RNA Affinity in Tandem technique (RAT), we identified novel<italic> nos </italic>translational repressors. Roles for these factors in translational control throughout oogenesis and embryogenesis will be further investigated by taking advantage of the genetic resources available in <italic>Drosophila</italic>, together with molecular and biochemical methods.