MITOCHONDRIAL RNA EDITING IN TRYPANOSOMA BRUCEI

布氏锥虫的线粒体 RNA 编辑

• 批准号: 2672218
• 负责人: DONNA Jay KOSLOWSKY
• 金额: $ 10.2万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672218
• 关键词: RNA binding protein; RNase protection assay; Trypanosoma; affinity chromatography; crosslink; gel electrophoresis; gel filtration chromatography; messenger RNA; mitochondria; northern blottings; nucleic acid structure; oligonucleotides; polyadenylate; polymerase chain reaction; posttranscriptional RNA processing; spliceosomes

The phenomenon of mitochondrial RNA editing in kinetoplastid protozoans is the most radical form of post transcriptional alteration known to date. The editing of messenger transcripts in these organisms can create over half of the mRNA by uridine insertions. It allows the generation of translatable messages by creating the open reading frames as well as proper initiation and termination signals. In Trypanosoma brucei, there is a transcript specific mechanism that regulates editing during the life cycle of the parasite. However, almost nothing is known about the mechanism of RNA editing or how editing might be developmentally regulated. The overall objective of this proposal is to identify the components of the editing mechanism and to determine the pathway and requirements of their assembly into an active complex. This project will be approached using three avenues of research: 1) An analysis of the proteins and/or RNAs which specifically associate with unedited mRNA transcripts. Approaches used in analysis of spliceosomes and polyadenylation complexes will be employed as models, including: native gel electrophoresis, UV crosslinking, gel filtration and affinity chromatography. The pathway and requirement of assembly of these components into an active complex will be investigated utilizing native gel electrophoresis and two partial in vitro editing assays. 2) A determination of the sequence and secondary structure requirements for gRNA selection and use. By manipulating input pre-edited mRNAs and gRNAs in partial in vitro editing assays, the minimum sequence and 2 degree structure requirements for gRNA recognition and function will be determined. In addition, different models for how guide RNAs interact with and direct the editing process will be tested. 3) Development of a complete in vitro editing system. An assay in which a full cycle of editing can be monitored is crucial to the identification of specific editing components and to the determination of their individual functions. Therefore, considerable effort will be invested in developing a functional assay. Members of the kinetoplastida are the causative agents of African sleeping sickness, Chagas disease and leishmaniasis. There are no vaccines against these organisms and present chemotherapies are quite toxic. kRNA editing is unique to these organisms, and offers a prime target for the development of new treatments. The ability of small RNAs to dramatically increase the coding capacity of another class of RNA offers strong support for the role of RNA in the origins of life. Understanding RNA editing in these organisms may lend considerable insight to the evolution of early replication mechanisms, and has substantial implications on our understanding of the storage and regulation of genetic information.

线粒体RNA编辑的现象在动力学原生动物中 是已知的转录后变化的最激进形式 日期。在这些生物体中的信使成绩单的编辑可以创建 超过一半的mRNA插入。它允许一代 通过创建开放式阅读帧以及可翻译消息的 适当的启动和终止信号。在布鲁氏锥虫瘤中，那里 是转录本特定机制，可调节在此期间进行编辑 寄生虫的生命周期。但是，几乎一无所知 RNA编辑的机制或如何开发编辑 受监管。该提议的总体目的是确定 编辑机制的组成部分，并确定途径和 它们的组装要求成一个主动的复合体。这个项目将 可以使用三个研究途径进行处理：1） 蛋白质和/或RNA专门与未编辑的mRNA相关 成绩单。用于分析剪接体和的方法 多腺苷酸化合症将被用作模型，包括：天然 凝胶电泳，紫外线交联，凝胶过滤和亲和力 色谱法。这些组装的途径和要求 将使用天然来研究进入活跃复合物的组件 凝胶电泳和两个部分体外编辑测定法。 2）a 确定序列和二级结构要求 选择和使用。通过操纵输入预编辑的mRNA和GRNA 在部分体外编辑测定中，最小序列和2度 GRNA识别和功能的结构要求将是 决定。此外，指导RNA相互作用的不同模型 使用和直接编辑过程将进行测试。 3）开发 完整的体外编辑系统。一个完整周期的测定 可以监视编辑对于识别特定的识别至关重要 编辑组件并确定其个人 功能。因此，将大量的努力用于开发 功能测定。动作层的成员是因果关系 非洲睡眠疾病，查加斯病和利什曼病的特工。 没有针对这些生物的疫苗，并进行化学疗法 很有毒性。 KRNA编辑是这些生物独有的，并提供 开发新疗法的主要目标。能力 小型RNA急剧增加另一类的编码能力 RNA的RNA对RNA在生命的起源中的作用提供了强有力的支持。 了解这些生物中的RNA编辑可能会大大借出 深入了解早期复制机制的发展，并具有 对我们对存储的理解和 遗传信息的调节。