GRNA/MRNA INTERACTIONS IN TRYPANSOME RNA EDITING

锥虫 RNA 编辑中的 GRNA/mRNA 相互作用

• 批准号: 6691091
• 负责人: DONNA Jay KOSLOWSKY
• 金额: $ 23.23万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6691091
• 关键词: Trypanosoma brucei rhodesiense; affinity labeling; complementary RNA; crosslink; gel electrophoresis; gel filtration chromatography; gel mobility shift assay; intermolecular interaction; messenger RNA; nucleic acid structure; posttranscriptional RNA processing; surface plasmon resonance

DESCRIPTION: RNA editing in Trypanosoma brucei is a process that inserts / deletes uridylate residues (U) from mitochondrial pre-mRNA molecules. This phenomenon produces mature mRNAs by creating open reading frames, correcting frame shifts and creating signals for translational initiation and termination. The placement of Us is guided by a small RNA molecule (the guide (g) - RNA) which is complementary to portions of the mature mRNA. While the gRNA is known the carry the information for the editing process, little is known about how the gRNAs are used to direct the editing process. All of the gRNAs identified to date have defined 5' anchor sequences, guiding sequences and a non-encoded 3' uridine tail. The overall objective of this proposal is to understand how the different gRNA elements contribute to the editing process, how they interact with the pre-edited mRNA during editing and ultimately how the gRNA is utilized to direct editing. This objective will be approached using three avenues of research: 1) an analysis of the secondary structure interactions between gRNA and mRNA during the editing process. The extent of the gRNAs interactions with the mRNA will be determined by combining photoaffinity cross linking with structure probing techniques. Defining the structures of the gRNA and the mRNA as they interact during the editing process will help lead to a molecular understanding for the role of the gRNAs in the editing process. 2) An analysis of the tertiary structure of RNAs and proteins in the editing complex. The cross linking patterns of photo-agents attached at homologous sites in different gRNAs and mRNAs will be analyzed to determine if these RNAs contain a common, core tertiary structure. Using site-specific cross linking techniques, the positions of editing proteins in relation to specific regions of the gRNA and mRNA will be analyzed. 3) A determination of the roles of sequence and structure in the kinetics of gRNA/mRNA association. The contribution both sequence and structure make in the overall kinetics of RNA interaction will be analyzed using native and temperature gradient gel electrophoresis and surface plasmon resonance technology. The binding of the gRNA to the mRNA is the fundamental step in RNA editing. An understanding of the nature and relative importance of the elements, which confer specificity on this interaction, is critical to our understanding of the editing process. This research will allow us to begin to understand the structural function relationship of RNAs and protein within the editing complex and greatly enhance our understanding of the mechanism involved in the transfer of information from one RNA molecule to another. Members of the kinetoplastida are the causative agents of African sleeping sickness, Chagas disease and leishmaniasis. Understanding kRNA editing, which is unique to these organisms, will contribute to the fundamental knowledge of the parasite and may lead to the development of new strategies for disease intervention and control.

描述：在布氏锥虫中的RNA编辑是一个插入/ 从线粒体前信使核糖核酸分子中删除尿苷酸残基(U)。这 现象通过创建开放阅读框架、纠正 帧移动，并产生翻译开始和结束的信号。 我们的定位是由一个小的RNA分子(GUIDE(G)-RNA)引导的 这与部分成熟的mRNA是互补的。虽然gRNA是已知的 携带编辑过程的信息，人们对此知之甚少 GRNA用于指导编辑过程。所有已鉴定的gRNA 到目前为止，已经定义了5‘锚定序列、引导序列和非编码序列 3‘-尿苷尾巴。这项建议的总体目标是了解如何 不同的gRNA元素对编辑过程有贡献，它们是如何 在编辑过程中与编辑前的mRNA相互作用，并最终确定gRNA的状态 用来指导编辑的。这一目标将通过三个方面来实现 研究途径：1)二级结构相互作用分析 在编辑过程中gRNA和mRNA之间的关系。GRNAs的范围 与mRNA的相互作用将通过结合光亲和交叉来确定 与构造探测技术相结合。确定gRNA的结构 在编辑过程中相互作用的mRNA将有助于导致 对gRNA在编辑过程中的作用的分子理解。2)一个 分析编辑复合体中的RNA和蛋白质的三级结构。 光敏剂在同源位点上的交联型式 将分析不同的gRNAs和mRNAs以确定这些RNAs是否包含 共同的、核心的三级结构。使用特定部位的交联链技术， 编辑蛋白质相对于gRNA特定区域的位置 并对信使核糖核酸进行分析。3)顺序和顺序角色的确定 结构在gRNA/信使核糖核酸结合的动力学中。这两方面的贡献 序列和结构在整个RNA相互作用动力学中的作用将是 使用自然和温度梯度凝胶电泳和表面分析 等离子激元共振技术。GRNA与mRNA的结合是 RNA编辑的基本步骤。对自然与相对关系的理解 赋予这种相互作用特殊性的元素的重要性是 对于我们对编辑过程的理解至关重要。这项研究将允许 美国开始了解RNA和RNA的结构功能关系 编辑复合体中的蛋白质并极大地增强了我们对 一种将信息从一个RNA分子转移到 又一个。动胞体的成员是非洲的病原体 昏睡病、恰加斯病和利什曼病。理解奎师那 编辑，这是这些有机体所独有的，将有助于基本 对寄生虫的了解，并可能导致制定新的应对策略 疾病干预和控制。