SPECIFICITY OF RNA MATURATION

RNA 成熟的特异性

• 批准号: 2177341
• 负责人: BARBARA SOLLNER-WEBB
• 金额: $ 20.35万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177341
• 关键词: SPECIFICITY; RNA; MATURATION

The overall goal of the research described in this grant application is to better understand a very bizarre and novel process of transcript maturation that involves the often very extensive insertion (and occasional deletion) of U residues from primary transcripts to achieve the mature RNA sequences. This "RNA editing" has thus far been shown to be utilized by mitochondrial RNAs of trypanosomes but it may well be used by other organisms as well and is thought to provide clues to the early evolution of RNA metabolism This U insertion/deletion is "guided" by short complementary RNAs (gRNAs) which evidently also serve to donate their U residues to the mRNA through formation of a chimeric intermediate with the mRNA portion downstream of the site of editing. But how these gRNAs do the guiding and what is the mechanism of formation of the chimeric intermediate and of the entire reaction are some of the crucial, unresolved questions about RNA editing that we plan to address. Using a trypanosome mitochondrial extract recently shown to form such gRNA/mRNA chimeras in vitro, we will investigate the mechanism/catalytic basis of this chimera formation. To help determine whether the in vitro chimera formation occurs by transesterification or by an endonuclease/ligase route, we will identify the precise site of the junction and the stereochemical considerations imposed on the junction phosphate of the chimer~ We will further analyze chimera formation by studying known co- fractionating activities and by further purification of the activity. We will also attempt to recreate an entire editing reaction in vitro (preliminary very encouraging results have now shown specific cleavage of the gRNA/mRNA chimera and reformation of an intact partly edited mRNA!) and then will study this reaction. Analysis in permeabiIized cells should provide much needed information about the kinetics of the editing process, including the production of the surprisingly abundant aberrant edited molecules. In complementary studies of the steady state mitochondrial RNA population, we will clone and analyze various intermediates in the editing process, asking whether these molecules correspond to predictions from transesterification or nuclease/ligase models of editing, whether editing occurs in strict 3'-> 5' order or shows some flexibility, and whether activities suggested from the in vitro analysis are utilized for editing in vivo. We will use in situ hybridization analysis to localize where within the trypanosome mitochondria the unedited, the partially edited, and possibly the fully edited RNAs are located. This information could allow determination of whether editing occurs in the kinetoplast mitochondrial body or in the long mitochondrial tubule that extends from it along the length of the trypanosome. Through these studies we hope to gain a better understanding about this very ancient and novel form of RNA maturation.

本赠款申请中描述的研究的总体目标是 更好地理解一个非常奇怪而新颖的成绩单过程 成熟涉及通常非常广泛的插入（和 偶尔的删除）来自主要转录本的U残基以实现 成熟的RNA序列。到目前为止，此“ RNA编辑”已被证明是 由锥虫的线粒体RNA使用，但很可能由 其他生物也被认为为早期提供线索 RNA代谢的进化此U插入/缺失是“指导”的 互补的RNA（grnas）显然也有助于捐赠他们 通过形成与嵌合中间的残留物 编辑站点下游的mRNA部分。但是这些grnas是如何做的 引导以及嵌合形成的机制是什么 中间和整个反应是至关重要的， 关于我们计划解决的有关RNA编辑的未解决的问题。使用 锥体线粒体提取物最近显示出形成这种GRNA/mRNA 嵌合体在体外，我们将研究 这个嵌合体的形成。帮助确定体外嵌合体是否 形成是通过移度或核酸内切酶/连接酶发生的 路线，我们将确定交界处的确切位置 立体化学考虑对磷酸盐的结 Chimer〜我们将通过研究已知共同的共同体进一步分析嵌合体的形成 分级活动和通过进一步净化活动。我们 还将尝试在体外重新创建整个编辑反应 （初步非常令人鼓舞的结果现已显示出特定的裂解 GRNA/mRNA嵌合体和完整的部分编辑mRNA的改革！） 然后将研究这一反应。透气性细胞中的分析应 提供有关编辑过程动力学的急需信息， 包括编辑令人惊讶的丰富异常的生产 分子。在稳态线粒体RNA的互补研究中 人口，我们将克隆并分析编辑中的各种中间体 过程，询问这些分子是否对应于 编辑的固定酶或核酸酶/连接酶模型是编辑 以严格的3' - > 5'订单发生或显示一定的灵活性，以及​​是 从体外分析中建议的活动用于编辑 体内。我们将使用原位杂交分析来本地化 在锥虫线粒体内，未经编辑的部分编辑，部分编辑， 并且可能位于完全编辑的RNA。这些信息可以 允许确定在动力学成形术中是否发生编辑 线粒体或在长的线粒体小管中，从 它沿着锥虫的长度。通过这些研究，我们希望 对这种非常古老和新颖的RNA的理解更好 成熟。