RNA Transport

小行星9601209 在20世纪80年代，随着特异性mRNA靶向爪蟾的给定区域的发现，开辟了一个新的研究领域（King和Barklis，1985; Rebagliati等人，1985）和果蝇卵母细胞（Lehamann和Nusslein-Volhard，1986; Driever和Nusslein-Volhard，1988）。 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，其确定包括头部、胸部和腹部（Wang和Lehmann，1991）以及生殖细胞谱系（Ephrussi和Lehmann，1992）的区域身份。 最近，在体细胞中已经描述了许多定位的mRNA，并且现在清楚的是，RNA靶向是定位特定蛋白质的重要调节机制（St.约翰斯顿，1995）。 然而，很少有人知道RNA是如何选择，运输和锚定在特定的亚细胞结构域。 我们的实验室已经分离并表征了七种RNA，它们在爪蟾卵母细胞中显示出两种基本的植物皮质定位途径（Forristall et al.，1995年）。 在这项提案中，重点将是分析Xcat-2和VegT RNA作为两种途径的代表，因为它们在早期发育过程中的重要性。 VegT编码一种可以调节其他基因活性的蛋白质。 它包含一个与小鼠短尾畸形（T基因）DNA结合结构域50%相同的结构域，短尾畸形是一种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.例如，1995年）。 在非洲爪蟾胚胎的腹侧注射VegT RNA诱导第二背轴。 VegT的错误表达导致无头胚胎（Zhang和King，1996 b）。 Xcat-2 RNA编码一种可能对生殖细胞决定和配子产生至关重要的RNA结合蛋白。 Xcat-2与包含生殖细胞的假定决定子的种质共定位。 King博士的工作假设是，RNA是根据其结构中3 'UTR中的定位信号以及特异性结合的蛋白质来选择转运的。 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中是保守的。 为了验证她的假设，她提出了以下具体目标：1。 确定Xcat-2和VegT的RNA定位信号和包含的功能元件。 2. 确定哪些蛋白质结合定位元件，并评估它们是否是定位所需的。 3. 确定在卵子发生过程中，植物定位的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在显微注射后定位于卵母细胞的植物皮质的能力。 将创建一系列RNA的缺失突变体，以鉴定定位信号和元件，并评估光交联蛋白对这些信号的功能。 最后，她将使用我们已经表征的其他七种定位的RNA来询问是否可以鉴定出共同的定位基序。 虽然卵母细胞将在这项研究中使用，因为它们提供了许多独特的优势，RNA定位的机制是最肯定的不同类型的细胞之间的保守。 因此，她的分析应该提供对细胞一般如何区域靶向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.