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MITOCHONDRIAL RNA EDITING IN TRYPANOSOMA BRUCEI

布氏锥虫的线粒体 RNA 编辑

基本信息

批准号：
2886835
负责人：
DONNA Jay KOSLOWSKY
金额：
$ 10.66万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 2001-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2886835
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

项目摘要

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.

动质体原生动物中的线粒体RNA编辑现象是已知的转录后改变的最根本形式，约会这些生物体中信使转录本的编辑可以创造超过一半的mRNA通过尿苷插入。它允许产生通过创建开放的阅读框架以及正确的启动和终止信号。在布氏锥虫中，是一种转录本特异性机制，在转录过程中调节编辑，寄生虫的生命周期然而，几乎没有人知道 RNA编辑的机制或编辑如何在发育过程中监管.本建议的总体目标是确定编辑机制的组成部分，并确定途径，将其组装成活性复合物的要求。该项目将可以通过三种研究途径进行：1）分析与未编辑的mRNA特异性结合的蛋白质和/或RNA 成绩单剪接体分析中使用的方法和多聚腺苷酸化复合物将被用作模型，包括：凝胶电泳、紫外交联、凝胶过滤和亲和层析层析组装的途径和要求将利用本机研究活性复合物中的组分凝胶电泳和两个部分体外编辑测定。2)一序列和二级结构要求的确定 gRNA选择和使用。通过操纵输入的预编辑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测定此外，指导RNA如何相互作用的不同模型使用并直接测试编辑过程。3)发展一个完整的体外编辑系统。一种测定法，其中一个完整的循环编辑可以被监测是至关重要的，以确定具体的编辑组件和确定其各自的功能协调发展的因此，将投入相当大的努力，功能测定。动质体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，毒性很强kRNA编辑是这些生物体所独有的，是开发新疗法的主要目标的能力小分子RNA能显著增加另一类RNA的编码能力这一发现为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编辑可能会提供相当大的帮助。洞察早期复制机制的演变，并已对我们理解存储和基因信息的调控。