GRNA/MRNA INTERACTIONS IN TRYPANSOME RNA EDITING

锥虫 RNA 编辑中的 GRNA/mRNA 相互作用

• 批准号: 6488735
• 负责人: DONNA Jay KOSLOWSKY
• 金额: $ 25.9万
• 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/6488735
• 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：布氏锥虫中的 RNA 编辑是一个插入 / 从线粒体前 mRNA 分子中删除尿苷酸残基 (U)。这 现象通过创建开放阅读框产生成熟的 mRNA，纠正 移码并产生翻译起始和终止信号。 Us 的位置由小 RNA 分子引导（引导 (g) - RNA） 它与成熟 mRNA 的部分互补。虽然 gRNA 已知 携带编辑过程的信息，但对其如何进行知之甚少 gRNA 用于指导编辑过程。所有已识别的 gRNA 迄今为止已经定义了5'锚定序列、引导序列和非编码序列 3' 尿苷尾。该提案的总体目标是了解如何 不同的 gRNA 元件对编辑过程有何贡献，它们如何 在编辑过程中与预编辑的 mRNA 相互作用以及最终 gRNA 的作用 用于直接编辑。该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 编辑过程中 gRNA 和 mRNA 之间的关系。 gRNA 的范围 与 mRNA 的相互作用将通过结合光亲和交叉来确定 与结构探测技术相联系。定义 gRNA 的结构 mRNA 在编辑过程中相互作用将有助于产生 gRNA 在编辑过程中的作用的分子理解。 2) 一个 分析编辑复合物中 RNA 和蛋白质的三级结构。 附着在同源位点的光剂的交联模式 将分析不同的 gRNA 和 mRNA，以确定这些 RNA 是否包含 共同的核心三级结构。使用位点特异性交联技术， 编辑蛋白质相对于 gRNA 特定区域的位置 并对 mRNA 进行分析。 3）确定顺序和角色的作用 gRNA/mRNA 关联动力学中的结构。贡献双方 RNA相互作用的整体动力学中的序列和结构将是 使用天然和温度梯度凝胶电泳和表面分析 等离子共振技术。 gRNA 与 mRNA 的结合是 RNA编辑的基本步骤。对本质和相对关系的理解 赋予这种相互作用特殊性的元素的重要性是 对于我们理解编辑过程至关重要。这项研究将允许 我们开始了解RNA的结构功能关系 编辑复合体中的蛋白质，极大地增强了我们对 信息从一个RNA分子转移到另一个RNA分子的机制 其他。动质体成员是非洲疟疾的病原体 昏睡病、恰加斯病和利什曼病。了解 kRNA 这些生物体所独有的编辑将有助于基本的 对寄生虫的了解可能会导致制定新的策略 疾病干预与控制。