RNA Transport

RNA运输

• 批准号: 9601209
• 负责人: Mary Lou King
• 金额: $ 30万
• 依托单位: University of Miami School of Medicine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-15 至 2000-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9601209&HistoricalAwards=false
• 关键词: RNA; Transport

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.

9601209金在20世纪80年代的S，一个新的研究领域开辟了，发现特定的mRNA针对非洲爪哇(King和Barkis，1985年；Rebagliati等人，1985年)和果蝇卵母细胞(Lehamann和Nusslein-Volhard，1986年；Driever和Nusslein-Volhard，1988年)的特定区域。事实证明，RNA本地化对发展的影响是深远的。在果蝇中，局部RNA的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和Lehmann，1991)以及生殖细胞谱系(Efrussi和Lehmann，1992)。最近，已经在体细胞中描述了一些定位的mRNAs，现在很清楚，RNA靶向是定位特定蛋白质的重要调控机制(St.Johnston，1995)。然而，关于RNA是如何被选择、运输到特定的亚细胞结构域并在其中锚定的，人们知之甚少。我们的实验室已经分离并鉴定了7个RNA，它们在非洲爪哇卵母细胞中显示了两条基本的植物皮质定位途径(Forristall等人，1995年)。在这项提案中，重点将分析XCAT-2和Vegt RNAs作为这两个途径的代表，因为它们在早期发育过程中具有重要意义。Vegt编码一种蛋白质，可以调节其他基因的活动。它包含一个结构域，与小鼠短臂病毒的DNA结合结构域(T基因)有50%的同源性，T基因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)。在非洲爪哇胚胎腹侧注射Vegt RNA可诱导出第二背轴。Vegt基因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，可能是生殖细胞决定和配子产生所必需的。XCAT-2与包含生殖细胞假定决定因素的种质共定位。金博士的工作假设是，RNA是根据其3‘UTR结构中的定位信号以及特定结合的蛋白质来选择进行运输的。她进一步假设，运输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。为了测试她的假设，她建议完成以下具体目标：1.确定XCAT-2和Vegt的RNA定位信号和包含的功能元件。2.确定哪些蛋白质与定位元件结合，并评估它们是否需要定位。3.利用相同的卵子发生途径，确定植物定位的mRNAs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质的能力。将创建一系列RNA缺失突变体来识别定位信号和元件，并评估光交叉蛋白对这些信号的功能。最后，她将使用我们已经表征的另外七个本地化RNA来询问是否可以确定一个共同的本地化基序。尽管本研究将采用卵母细胞，因为它们提供了许多明显的优势，但RNA定位的机制在不同类型的细胞中几乎是保守的。因此，她的分析应该能让我们深入了解细胞一般是如何以RNA为靶标的。