RNA PROCESSING AND NUCLEAR RIBONUCLEOPROTEIN

我的研究计划的长期目标是了解 前mRNA加工事件的机制和控制， 特别强调RNA相关的 蛋白质（hnRNP和snRNP）。 用一种不寻常的方法 问题，“米勒”染色质扩散技术的电子 在显微镜下，我们已经可视化了剪接体的过程 在特定的新生转录物上形成。 似乎 剪接位点的选择发生得很早， 在RNP组件上使用拼接接头信号。 研究 本文所概述的将产生用于研究 RNA加工的RNP模板，使用果蝇作为 实验生物 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 研究-（a）将质粒载体上的基因显微注射到 果蝇胚胎，和（B）果蝇胚胎的种系转化 果蝇的基因连接到一个扩增控制元件- 这两种方法都导致了多拷贝，超微结构上可区分的 基因. 这些基因将在体外被操纵， 剪接信号的邻近性的重复或改变。 我们将 然后分析剪接体组装的一些要求， 在体内，剪接体形成的动力学在不同的 内含子，以及转录顺序的影响， 剪接位点序列在剪接点配对上的接近度。 免疫EM分析将定位特定的snRNP和hnRNP RNP复合物上的蛋白质。 最后，我们将开始克隆 果蝇hnRNP蛋白基因。 这将使（a） 与高等真核生物中的这些蛋白质进行进化比较， (b)一种获得果蝇蛋白抗体的方法， 免疫EM研究，和（c）基因测试的功能， proteins.

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.