The expression of ribosomal protein genes during Xenopus oogenesis and embryogenesis is regulated at the translational level and provides an excellent example of a developmental process amenable to analysis at the molecular level. This proposal focuses on three major aspects of the structure, regulation, and function of these genes. First, the factors that constitute the deadenylation activity which is required for the translational inactivation of maternal ribosomal protein mRNAs during oocyte maturation will be purified. In vitro systems will be used in order to biochemically characterize the deadenylation activity, its mechanism of poly(A) removal, and to determine its regulation during maturation and embryogenesis will be analyzed both in vivo and in vitro. These studies will further delineate cis-acting elements located in the 5' untranslated region of the ribosomal protein L1 mRNA that are required for this regulation. A cell-free extract derived from embryos retains the proper translational regulation of ribosomal protein mRNAs in vitro. This system will be used to identify transacting proteins involved in translational control and to determine the specificity of their interactions with ribosomal protein mRNAs. The role of translation initiation factor 4E in the control of ribosomal protein synthesis during embryogenesis will be ascertained. Third, functional domains within ribosomal protein L5 that are responsible for 5S rRNA binding, nuclear and nucleolar localization, and 60S subunit assembly will be identified. Biochemical and immunological assays will be used to determine the interrelationships among the regions of this protein responsible for these aspects of its function. These analyses will be undertaken in vivo by the microinjection of cloned ribosomal protein genes and systematically altered versions of these genes and gene products into oocytes and fertilized eggs. Parallel studies in vitro will use cell-free systems derived from oocytes and embryos at appropriate developmental stages to reconstitute translational control. These studies will provide basic information on specific RNA-protein interactions that are required for the coordinate control of constitutively expressed structural genes during normal development and the biosynthesis of the eukaryotic ribosome.
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