RNA Transport

在20世纪80年代，研究开辟了一个新的领域，发现特定的mrna针对爪蟾（King and Barklis, 1985; Rebagliati et al., 1985）和果蝇卵母细胞（Lehamann and Nusslein-Volhard, 1986; driver and Nusslein-Volhard, 1988）的特定区域。RNA定位对发育的影响已被证明是深远的。在果蝇中，局部RNA表达导致蛋白质梯度，决定了包括头部、胸部和腹部在内的区域身份（Wang and Lehmann, 1991）以及生殖细胞谱系（Ephrussi and Lehmann, 1992）。最近，在体细胞中描述了一些定位mrna，现在很清楚，RNA靶向是定位特定蛋白质的重要调节机制（St. Johnston, 1995）。然而，关于rna是如何被选择、转运到特定的亚细胞结构域和锚定的，我们知之甚少。我们的实验室已经分离并鉴定了七种rna，它们在爪蟾卵母细胞中显示出两种基本的植物皮层定位途径（Forristall etal ., 1995）。在本提案中，重点将分析Xcat-2和VegT rna作为这两种途径的代表，因为它们在早期发育过程中很重要。VegT编码一种可以调节其他基因活动的蛋白质。它包含一个与小鼠brachyury （T基因）DNA结合域50%相同的结构域，brachyury是正常早期发育所需的转录因子，在其他脊椎动物中高度保守（Kisper和Herrmann, 1993; Kispert et. al., 1995）。在爪蟾胚胎的腹侧注射VegT RNA诱导出第二背轴。VegT的错误表达导致无头胚胎（Zhang和King， 1996b）。Xcat-2 RNA编码一种RNA结合蛋白，可能对生殖细胞的决定和配子的产生至关重要。Xcat-2与含有生殖细胞的推定决定因素的种质共定位。King博士的工作假设是，rna是根据其3'UTR结构内的定位信号以及特异性结合蛋白来选择运输的。她进一步假设，转运颗粒的组成部分以及RNA定位基序可能在使用相同途径的植物定位RNA中保守。为了验证她的假设，她建议实现以下具体目标：1。鉴定Xcat-2和VegT的RNA定位信号和包涵功能元件。2. 确定哪些蛋白质与定位元件结合，并评估它们是否为定位所必需。3. 为了确定在植物中使用相同途径定位的mrna之间是否存在共同的定位基序。她的方法利用了体外合成的rna在显微注射后定位到卵母细胞的植物皮层的能力。将创建一系列的rna缺失突变体来识别定位信号和元件，并评估光交联蛋白对这些信号的功能。最后，她将使用我们已经鉴定的其他七个定位rna来询问是否可以确定一个共同的定位基序。虽然本研究将使用卵母细胞，因为它们具有许多独特的优势，但RNA定位的机制在不同的细胞类型中肯定是保守的。因此，她的分析应该提供对细胞如何在一般区域靶向rna的见解。

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.