| The subcellular localization of mRNA is an efficient way to restrict protein synthesis to specific cytoplasmic areas, and serves to establish cell polarity, embryonic pattern and synaptic plasticity in organisms from flies to humans. Progress has been made in identifying cis-acting localization elements (LEs), trans-acting proteins and molecular motor proteins. Staufen is one trans-acting protein conserved from flies to man and has multiple roles in the localization of distinct mRNAs in diverse cell types. While one Staufen gene exists in flies, two homologous Staufen genes are identified in vertebrates.;In Xenopus oocytes, a number of maternai transcripts important for early embryonic development are sorted to the vegetal cortex. To understand further the mechanism of such mRNA vegetal localization, I cloned Xenopus Staufenl and Staufen2 mRNAs from an oocyte cDNA library and generated antibodies for both homologs. Based on biochemical assays, immunocytochemical methods, and functional assays performed by another laboratory, I concluded that xStaufen2 protein is not expressed at detectable levels in oocytes and xStaul is the major functional homolog for RNA localization.;While Staufen is thought to recognize double stranded RNAs, no one had yet identified any potential vertebrate RNA structure as a specific binding site for Staufen. Comparatively analyzing the potential RNA structures of the localization element of Xcat2 (MCLE), I hypothesized the intermolecular homodimer of MCLE may be important for Xcat-2 localization. A series of mutants of MCLE RNAs were analyzed using quantitative localization assays, and the results support a functional role of intermolecular RNA base-pairing for mRNA localization. This is the first example of such a structure in vertebrates.;Finally, RNA localization is thought in close association with the endoplasmic reticulum. During analysis of Staufen function I discovered that RNase treatment of oocytes extract before subcellular fractionation released xStaul, Vera and 40LoVe from the ER-enriched fractions, suggesting their association with the ER depends on RNAs. On the contrary, CPEB 1 became concentrated in the ER-enriched fractions after RNase treatment. This and several other observations suggest a model in which the CPEB 1 protein, and possibly the CPEB1 mRNA, facilitate the association of localized RNAs with the endoplasmic reticulum. |
