): Embryonic development involves cell fate specification, cell division, and cell differentiation. These events begin with the localization of regulatory molecules, including RNAs, in the egg. The PI has used ascidian eggs as a model system to investigate localized RNAs. Ascidians are simple chordates with advantages for studying developmental processes, including eggs with colored regions of specific developmental fate (e.g., the yellow crescent or myoplasm) and closely related species with different modes of development (e.g., tailed and tailless larva). Using these attributes, the PI has identified the yellow crescent (YC) and Uro-1 RNAs, which are localized in the myoplsam (muscle-forming region), and proliferating cell nuclear antigen (PCNA) mRNA, which is localized in the ectoplasm (epidermal and neural-forming region) of ascidian eggs. The YC and PCNA RNAs have complementary 3 UTRs and may represent a novel means for controlling the extent of embryonic cell division by antisense RNA. Uro-1 is an ankrin repeat-containing (SHARK family) protein tyrosine kinase (PTK), which is localized in the myoplsam and required for muscle differentiation. The objective is to determine the functions of these localized RNAs in development. The first specific aim is to map the YC/PCNA locus by sequencing genomic clones and to determine whether YC RNA codes for a protein. The second specific aim is to determine the expression patterns of YC and PCNA RNA during development by blot and in situ hybridization. The third specific aim is to investigate the role of YC/PCNA RNA interactions in PCNA mRNA stability and translation using in vivo assays and by 3 UTR swapping experiments. The fourth specific aim is to investigate the function of YC and PCNA RNA by gain- and loss-of-function studies using synthetic mRNAs and antisense oligos. The fifth specific aim is to determine the subcellular localization of Uro-1 PTK using antibodies. The sixth specific aim is to identify proteins associated with the Uro-1 PTK by immunoprecipitation and two-hybrid system cloning. The seventh specific aim is to determine the developmental function of Uro-1 using antisense oligos and synthetic mRNAs. This investigation will provide new information on the regulation of cell division and cell fate determination during embryogenesis.
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