Immuno-ultrastructural Analysis of the Splicing Complex

该实验室使用一种不同寻常的体内超微结构方法“米勒染色质铺展技术”，在电子显微镜下显示转录活性基因，并分析新生前mRNA转录物上的共转录RNA加工事件，包括核糖核蛋白(RNP)的形成、剪接位点选择、构成剪接和替代剪接。为了将观察从超微结构现象学水平扩展到特定蛋白质的生化鉴定，需要能够识别转录本上特定位置和特定结构中的蛋白质。免疫电子显微镜技术可以进行这种分析，但还没有完善到用于米勒染色质铺展。拟议的项目将对这种技术的开发采取系统和合乎逻辑的方法。一些所需的单抗已经问世，将与胶体金标记物一起用于EM可视化。这种技术的即时应用包括在新生RNA上定位特定的SnRNP、特定的hnRNP蛋白质、一般剪接因子和参与剪接调控的蛋白质。然而，这项技术将更普遍地有用，并可用于将涉及染色质结构、复制、转录调节、衰减和多腺苷基化的蛋白质定位到遗传物质上的特定位置或结构。因此，这项技术的发展将使人们有可能更深入地了解蛋白质如何与DNA和RNA相互作用，以影响和调节基因表达。

This laboratory uses an unusual in vivo ultrastructural approach, the "Miller chromatin spreading technique," to visualize transcriptionally active genes in the electron microscope and to analyze the co-transcriptional RNA processing events on nascent pre-mRNA transcripts, including ribonucleoprotein (RNP) formation, splice site selection, constitutive splicing, and alternative splicing. In order to extend observations from the level of ultrastructural phenomenology to biochemical identification of specific proteins, there is a need to be able to identify the proteins at specific sites on the transcripts and in specific structures. The technique of immuno-electron microscopy would allow this analysis, but has not yet been perfected for use on Miller chromatin spreads. The proposed project will take a systematic and logical approach to the development of such a technique. Some of the desired monoclonal antibodies are already in hand, which will be used in conjunction with colloidal gold markers for EM visualization.Immediate applications of such a technique include localization on nascent RNA of specific snRNPs, specific hnRNP proteins, general splicing factors, and proteins implicated in splicing regulation. The technique would be more generally useful, however, and could be used to localize proteins involved in chromatin structure, replication, transcription regulation, attenuation, and polyadenylation to specific sites or structures on the genetic material. The development of this technique would thus make possible a deeper understanding of how proteins interact with DNA and RNA to effect and regulate gene expression.