A Chimeric mRNA for Studying Localization and Translational Control in Xenopus Oocytes

Brendan D. Crawford
University of Notre Dame

Localization of certain maternal mRNAs is necessary for proper differentiation of developing oocytes and embryos. Vg1 mRNA localizes to the vegetal hemisphere of Xenopus laevis oocytes. It encodes a member of the TGF-ƒÒ family of proteins that is necessary for induction of dorsal mesoderm. In the 3¡¦ untranslated region, a 340-nucleotide region called the vegetal localization element (VLE) directs localization while a 250-nucleotide region called the translational control element (TCE) mediates translational repression of Vg1 mRNA until localization is complete. Approximately half a dozen proteins bind to the VLE. Currently, the identity of five of these trans-acting factors has been determined; however, the individual functions of these proteins in localization are unknown. I have constructed a plasmid for the in vitro synthesis of mRNA that contains the 5¡¦ and 3¡¦ untranslated regions of Vg1 mRNA flanking the coding sequence for red fluorescent protein (RFP). The sequence encoding RFP can serve as a unique target for in situ hybridization in order to monitor the localization of injected mRNA. Concomitantly, the intrinsic red fluorescence of the protein provides a means to monitor the translational control of the mRNA in live oocytes. The mRNAs synthesized from this construct and its variants, missing the VLE or the TCE, will provide a valuable tool for studying the relationship between localization and translational control as well as the function of the trans-acting factors that bind to the VLE.