NSF Award
9601209
9601209 King In the 1980's, a new area of investigation opened up with the discovery that specific mRNAs are targeted to given regions of the Xenopus (King and Barklis, 1985; Rebagliati et al., 1985) and Drosophila oocyte (Lehamann and Nusslein-Volhard, 1986; Driever and Nusslein-Volhard, 1988). The consequences of RNA localization for development have proven to be profound. In Drosophila, local RNA expression results in protein gradients that determine regional identity including head, thorax, and abdomen (Wang and Lehmann, 1991) as well as the germ cell lineage (Ephrussi and Lehmann, 1992). More recently, a number of localized mRNAs have been described in somatic cells, and it is now clear that RNA targeting is an important regulatory mechanism for localizing particular proteins (St. Johnston, 1995). However, little is known about how RNAs are selected for, transported to and anchored in specific subcellular domains. Our lab has isolated and characterized seven RNAs which display two basic localization pathways to the vegetal cortex in Xenopus oocytes (Forristall et al., 1995). In this proposal, the focus will be on analyzing Xcat-2, and VegT RNAs as representatives of the two pathways because of their importance in early developmental processes. VegT encodes a protein that can regulate the activity of other genes. It contains a domain that is 50% identical with the DNA binding domain of mouse brachyury (T gene), a transcription factor required for normal early development and highly conserved in other vertebrates (Kisper and Herrmann, 1993; Kispert et. al., 1995). VegT RNA injection on the ventral side of Xenopus embryos induces a second dorsal axis. Mis-expression of VegT results in headless embryos (Zhang and King, 1996b). Xcat-2 RNA encodes an RNA binding protein that may be essential for germ cell determination and gamete production. Xcat-2 co-localizes with the germ plasm which contains the presumptive determinants for the germ cells. Dr. King's working hypothe sis is that RNAs are selected for transport based on localization signals within their structure in the 3'UTR, together with specifically bound proteins. She further hypothesizes that the component parts of the transport particle as well as the RNA localization motif are likely to be conserved among vegetally localized RNAs using the same pathway. To test her hypostheses, she proposes to accomplish the following specific aims: 1. To identify the RNA localization signal and inclusive functional elements for Xcat-2 and VegT. 2. To determine what proteins bind the localization elements and to assess whether they are required for localization. 3. To determine if there is a common localization motif among vegetally localized mRNAs using the same pathway in oogenesis. Her approach takes advantage of the ability of in vitro synthesized RNAs to localize to the oocyte's vegetal cortex after microinjection. A series of deletion mutants of the RNAs will be created to identify localization signals and elements and to assess the function of photocrossinked proteins to these signals. Finally, she will use seven other localized RNAs that we have characterized to ask if a common localization motif can be identified. Although oocytes will be employed in this study because they offer many distinct advantages, the mechanism of RNA localization is most certainly conserved among diverse cell types. Therefore, her analysis should provide insight into how cells in general regionally target RNAs.
