CAREER: Nuclear RNA Processing, Coilin and Cajal Bodies

分子事件，如RNA转录和成熟，如何影响核结构是真核细胞核的一个重要生理方面。近年来，这一中心问题引起了人们对原子核组织的新兴趣，并重新发现了几个离散的核细胞器。本研究项目的总体目标是评估其中一个核细胞器--卡哈尔小体(CB)在调控核RNA加工中的作用。使用的主要实验系统是非洲爪哇卵母细胞的核，因为它提供了以无与伦比的空间分辨率来探讨这一问题的独特机会。将讨论两个基本问题：通过CBS运输的小核核糖核蛋白颗粒(SnRNPs)的功能相关性是什么，以及CBS的标志性蛋白Colin如何将SnRNPs招募到CBS？柯林将作为一种分子工具，在不影响CBS表观生理的情况下，阻止SNRNPs进入CBS。它将允许在体内研究是否需要通过CBS的SnRNPs流动来使其成熟和/或处理前信使RNA的某些方面。还将对卵母细胞提取液进行生化分级，以确定可能与Colin相互作用的核质蛋白和RNA。然后，这些信息将被用于各种细胞系统，以确定柯林和新发现的因子将SNRNP招募到CBS的分子机制。研究柯林的生化和细胞动力学与SNRNPs的关系将为深入了解CBS的核作用提供新的见解。由于它的视觉吸引力和可访问性，这项研究非常适合积极的外展计划。它将通过指导一名高中生物教师和班级，并向包括女性和少数族裔在内的许多本科生和社区大学学生介绍生物学和新成像技术的奇迹，将其应用于几个教育级别。

How molecular events, such as RNA transcription and maturation, influence nuclear architecture is an important physiological aspect of the eukaryotic cell nucleus. In recent years this central question promoted renewed interest in the organization of the nucleus and rediscovery of several discrete nuclear organelles. The overall objective of this research project is to evaluate the role of one of these nuclear organelles, the Cajal body (CB), in the regulation of nuclear RNA processing. The main experimental system used is the nucleus of Xenopus laevis oocytes, because it provides the unique opportunity to approach this question with unequaled spatial resolution. Two essential questions will be addressed: What is the functional relevance of small nuclear ribonucleoprotein particles (snRNPs) trafficking through CBs, and how does coilin, the signature protein of CBs, recruit snRNPs to CBs? Coilin will be used as a molecular tool to prevent the entry of snRNPs into CBs, without affecting the apparent physiology of the latter. It will allow the investigation in vivo of whether the flow of snRNPs through CBs is required for some aspects of their maturation and/or the processing of pre-messenger RNAs. Biochemical fractionations will also be performed on oocyte extracts to identify the putative nucleoplasmic proteins and RNAs that interact with coilin. This information will then be used in various cell systems to define the molecular mechanisms by which coilin, and the newly identified factors, recruit snRNPs to CBs. Investigation of coilin's biochemistry and cellular dynamics in relation with snRNPs will provide new insights into the nuclear roles of CBs. Because of its visual appeal and accessibility, this research is highly amenable to an active outreach program. It will be applied at several educational levels by mentoring a high school biology teacher and class, and introducing many undergraduate and community college students, including women and minorities, to the wonders of biology and new imaging technologies.