DETERMINANTS OF SPECIFICITY IN KINETOPLASTID RNA EDITING

动质体 RNA 编辑特异性的决定因素

• 批准号: 6149938
• 负责人: Robert P Igo
• 金额: $ 3.75万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6149938
• 关键词: SDS polyacrylamide gel electrophoresis; Trypanosoma brucei rhodesiense; autoradiography; enzyme activity; gene expression; high performance liquid chromatography; mitochondria; nucleic acid sequence; nucleic acid structure; nucleotides; polymerase chain reaction; posttranscriptional RNA processing; protein purification; transposon /insertion element; uridine monophosphate

Kinetoplastid RNA editing inserts and deletes uridylate (U) residues in mitochondrial pre-mRNAs of trypanosomatids to produce mature mRNA. Small guide RNAs (gRNAs) specify the edited sequence. RNA editing proceeds by a mechanism involving endonucleolytic cleavage of pre-mRNA, addition or removal of Us at the 3' end of the 5' cleavage product, and finally, ligation of the cleavage products. The details of this process are still unclear; it is not certain how sequence information is transferred from gRNA to pre-mRNA. The goal of the proposed research is to elucidate the precise mechanism by which the edited sequence is determined. The primary research tools used will be an in vitro assay for RNA editing developed previously, and a variation, the precleaved editing substrate, in which the pre-mRNA is introduced as two cleavage fragments. To examine the role of guiding nucleotides in insertion editing, gRNAs will be mutated such that they can no longer serve as an RNA template for U addition, to see whether they can still guide U addition. The ability of nucleotides other than U to be added to a 5' cleavage product, in an editing context, will be assessed. Secondly, the contributions of the U-addition/removal and RNA ligation steps in specifying the number of Us inserted or deleted will be measured, using precleaved editing assays under reaction conditions allowing only one step to proceed. Finally, regions of complementarity between gRNA and pre-mRNA will be disrupted or created to determine the importance and characteristics of RNA-RNA duplex structures in editing. A thorough understanding of the mechanism of RNA editing in trypanosomatids may lead to novel therapeutic strategies against these parasites, and measures to stop their propagation by disrupting their cyclic transmission between insect and mammalian hosts.

动力质体RNA编辑插入物和缺失尿苷（U）残基（U）残基 锥形的线粒体前骨NAS产生成熟的mRNA。 小引导RNA（GRNA）指定编辑的序列。 RNA编辑 通过涉及前mRNA的内核解水解裂解的机制进行收益， 在5'切割产品的3'末端增加或删除我们，并且 最后，裂解产品的连接。 此过程的详细信息 仍然不清楚；不确定序列信息如何 从GRNA转移到前MRNA。 拟议研究的目标 是为了阐明编辑序列为 决定。 所使用的主要研究工具将是体外测定 对于先前开发的RNA编辑和一个变化， 编辑底物，其中将前MRNA作为两个裂解引入 碎片。 检查引导核苷酸在插入中的作用 编辑，grnas将被突变，以至于他们再也不能用作 添加u的RNA模板，看看它们是否仍然可以指导u 添加。除u以外的核苷酸的能力添加到5' 在编辑环境中的切割产品将被评估。其次， U型/删除和RNA连接步骤的贡献 将测量指定插入或删除的我们的数量，使用 在反应条件下排除的编辑测定只允许一个 步骤继续。 最后，GRNA和 前MRNA将被破坏或创建以确定重要性和 RNA-RNA双链结构在编辑中的特征。 彻底 了解锥形剂中RNA编辑机理的理解可能 导致针对这些寄生虫的新型治疗策略，并 通过破坏循环的措施来停止传播的措施 昆虫和哺乳动物宿主之间的传播。