The main focus of this research is to study RNA localization in leech embryos and to understand its role in normal embryonic development. RNA localization provides a mechanism for cells to specify the correct location for protein synthesis. RNA localization has been identified in embryonic and adult cells, and in organisms such as yeast, and several invertebrate, and vertebrate species. RNA localization, regardless of the type of cell or species, has several features in common. It requires the interaction between RNA-binding proteins and specific mRNA sequences, mainly on its 3'untranslated region (UTR). The transport and anchorage of mRNA is dependent on the cytoskeleton. In many organisms studied, the 3'UTR contains specific sequences involved in anchorage, transcriptional stability, translational regulation, and localization. All four functions (stability, translational regulation, localization, and anchorage) are important, but it is not clear if they affect each other. The long-term objective of the proposed research is to understand how mRNA translational repression/derepression, and stability are used by cells, to regulate proper mRNA localization and anchorage. The specific aims of this pilot project are to functionally characterize localization and anchorage sites found in the Hro-Twist (leech transcript) 3'UTR. Four approaches will be used. First, GFPHro- Twist 3'UTR deletion constructs will be obtained to verify preliminary studies. Second, these clones will be transcribed in vitro and injected in the zygote, to test for proper mRNA localization and mRNA anchorage in vivo. Third, a LacZ/Hro-Twist-3'UTR reporter construct will be used, to test prospective sequences involved in stability regulation. Fourth, PCR mutagenesis of ACE2 and ARE2 elements will be performed, to find those which are critical for function. Based on preliminary studies, we hypothesize that ACE2 elements in the Hro-Twist coding region are required to localize this transcript to the CD cell, and that ARE2 elements found in the 3'UTR are required for anchorage to the teloplasm.