RNA PROCESSING AND NUCLEAR RIBONUCLEOPROTEIN

我的研究计划的长期目标是了解 前信使核糖核酸加工事件的机制和控制 特别强调与RNA相关的 蛋白质(hnRNPs和SnRNPs)。使用一种不同寻常的方法来实现 问题，电子的“米勒”染色质扩散技术 显微镜下，我们已经可视化了剪接体的过程 在特定新生转体上的体内形成。看起来， 剪接位点的选择很早就发生了，并通过相互作用 在具有拼接连接信号的RNP组件上。这项研究 这里概述的将产生一个可操作的系统来研究 用于RNA处理的RNP模板，使用果蝇作为 实验生物体。我们将首先为EM开发一个系统 特定活性基因的可视化。两种方法将是 调查--(A)将质粒载体上的基因显微注射到 果蝇胚胎，以及(B)种系转化 携带与扩增控制元件相关基因的果蝇- 两者都导致了多拷贝，在超微结构上可区分 基因。这些基因将在体外被操纵以产生 拼接信号附近的复制或变化。我们会 然后分析了剪接体组装的一些要求 体内剪接体形成的动力学研究 内含子，以及转录和转录顺序的影响 剪接连接配对上的剪接位点序列的邻近性。 免疫EM分析将定位特定的SNRNP和hnRNP RNP复合体上的蛋白质。最后，我们将开始克隆 编码果蝇hnRNP蛋白的基因。这将允许(A) 与高等真核生物中这些蛋白质的进化比较， (B)获得果蝇蛋白抗体的方法 免疫电子显微镜研究，以及(C)基因测试，以确定 蛋白质。

The long term goals of my research program are to understand the mechanisms and control of pre-mRNA processing events, with particular emphasis on the role played by RNA-associated proteins (hnRNPs and snRNPs). Using an unusual approach to this problem, the "Miller" chromatin spreading technique for electron microscopy, we have visualized the process of spliceosome formation in vivo on specific nascent transcipts. It appears that splice site selection occurs very early and through the interaction on RNP components with splice junction signals. The research outlined herein will yield a manipulatable system for the study of the RNP template for RNA processing, using Drosophila as the experimental organism. We will first develop a system for the EM visualization of specific active genes. Two approaches will be investigated - (a) microinjection of genes on plasmid vectors into Drosophila embryos, and (b) germline transformation of Drosophila with genes linked to an amplification control element - both resulting in multicopy, ultrastructurally-distinguishable genes. The genes will be manipulated in vitro to yield duplications or changes in proximity of splicing signals. We will then analyze some of the requirements for spliceosome assembly in vivo, the kinetics of spliceosome formation on different introns, and the influence of the order of transcription and proximity of splice site sequences on splice junction pairing. Immuno EM analysis will locate specific snRNPs and hnRNP proteins on the RNP complex. Lastly, we will begin to clone the genes encoding Drosophila hnRNP proteins. This will allow (a) an evolutionary comparison to these proteins in higher eukaryotes, (b) a means to obtain antibodies to the Drosophila proteins for immuno EM studies, and (c) a genetic test for the function of the proteins.