Identification and characterization of novel components of a localized mRNP in Drosophila

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Body axis formation in Drosophila melanogaster (Dm) requires the asymmetric deposition of maternal mRNAs in the oocyte. Among the best-characterized examples of localized transcripts are oskar and bicoid mRNAs, the localization of which defines antero-posterior polarity. mRNA localization in the cytoplasm depends on the nuclear history of the transcript. Localizing mRNPs assemble during transcription in the nucleus, where a core set of proteins binds to the maturing mRNAs. These proteins provide a platform for the formation of larger, dynamic assemblies in the cytoplasm that regulate mRNA transport, silencing and localized translation. One core component of spliced mRNPs is the exon junction complex (EJC), an RNA-binding complex that is known to recruit other proteins and protein complexes and that is required for localization of oskar mRNA. The EJC is conserved among metazoans and therefore constitutes an ideal model system to understand the mechanisms of RNA localization both in flies and in higher eukaryotes, where this process is thought to be essential in brain development. In Dm, only a few binding partners of the EJC have been identified and the exact molecular composition, dynamics and regulation of EJC-containing mRNPs is poorly understood. It is also unclear which interaction partners render the EJC transcript-specific. Another mRNP component present on both bicoid and oskar mRNPs is the protein Exuperantia (Exu), required for the localization of bicoid RNA. Our work on the structure and function of Exu suggests that association of Exu with an unknown binding partner might confer transcript-specificity. To date, no systematic biochemical studies on the purified native oskar and bicoid mRNPs have been performed to identify all components. However, to reconstitute higher order assemblies from recombinant proteins for functional and structural studies, an inventory of the proteins involved is critically important. Our aim is to biochemically isolate localized mRNPs from oocytes of transgenic flies using a customized TAP-tagging approach. We will then use mass spectrometry to identify novel components. We will map, in detail, the interactions between mRNP components in vitro and reconstitute core protein/RNA complexes from recombinant proteins. These will be a starting point for further structure-function studies.