CAREER: Nuclear RNA Processing, Coilin and Cajal Bodies

RNA转录和成熟等分子事件如何影响核结构是真核细胞细胞核的一个重要生理方面。近年来，这个中心问题重新引起了人们对细胞核组织的兴趣，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这些核细胞器之一，Cajal体（CB）在核RNA加工调控中的作用。使用的主要实验系统是非洲爪蟾卵母细胞的细胞核，因为它提供了独特的机会，以无与伦比的空间分辨率来解决这个问题。将解决两个基本问题：小核核糖核蛋白颗粒（snRNPs）通过CBs运输的功能相关性是什么，以及CBs的特征蛋白coilin如何将snRNPs招募到CBs？Coilin将被用作阻止snRNPs进入CBs的分子工具，而不会影响后者的表观生理。它将允许在体内研究snRNPs通过CBs的流动是否需要它们成熟和/或前信使rna加工的某些方面。还将对卵母细胞提取物进行生化分离，以鉴定可能与coilin相互作用的核质蛋白和rna。这些信息随后将用于各种细胞系统，以确定卷曲蛋白和新发现的因子将snRNPs招募到CBs的分子机制。研究与snRNPs相关的coilin生物化学和细胞动力学将为cb的核作用提供新的见解。由于其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。它将通过指导高中生物教师和班级，并向包括女性和少数民族在内的许多本科生和社区大学生介绍生物学的奇迹和新的成像技术，在几个教育层面得到应用。

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.