MITOCHONDRIAL RNA EDITING IN TRYPANOSOMA BRUCEI

动质体类原生动物线粒体RNA编辑现象 是已知的最激进的转录后改变形式 约会。编辑这些生物体中的信使转录可以创建 超过一半的信使核糖核酸插入尿苷。它允许一代人 通过创建开放阅读框架以及 正确的启动和终止信号。在布氏锥虫中，有 是一种特定于文字记录的机制，用于在 寄生虫的生命周期。然而，人们对此几乎一无所知。 RNA编辑的机制或编辑可能如何发展 受监管的。这项建议的总体目标是确定 编辑机制的组件，并确定路径和 将它们组装成活性复合体的要求。这个项目将 采用三种研究途径：1)分析 与未编辑的信使核糖核酸特异结合的蛋白质和/或RNA 成绩单。剪接体和剪接体分析中使用的方法 将使用聚腺苷酸化复合体作为模型，包括：天然 凝胶电泳、紫外光交联、凝胶过滤和亲和力 层析法。它们组装的途径和要求 活性复合体中的成分将利用原生物质进行研究 凝胶电泳法和两种部分体外编辑试验。2)A 序列和二级结构要求的确定 GRNA的选择和使用。通过操作输入的预编辑的mRNA和gRNA 在部分体外编辑试验中，最小序列和2度 GRNA识别和功能的结构要求将是 下定决心。此外，指导RNA如何相互作用的不同模型 与和导演的编辑过程将受到测试。3)发展 一个完整的体外编辑系统。一种化验方法，在这种化验方法中， 可监控性编辑是识别特定对象的关键 编辑组件及其各自的确定 功能。因此，将投入相当大的努力来开发 一种实用的分析。动质体纲的成员是致病的 非洲昏睡病、恰加斯病和利什曼病的病原体。 目前还没有针对这些微生物的疫苗，也没有目前的化疗方法 都是非常有毒的。Krna编辑是这些生物独有的，并提供 这是开发新疗法的首要目标。的能力 小RNA可显著提高另一类的编码能力 对RNA的研究为RNA在生命起源中的作用提供了强有力的支持。 了解这些生物体中的RNA编辑可能会给我们带来很大的帮助 对早期复制机制的演变的洞察，并 对我们对存储和 对遗传信息的监管。

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.