Leucyl-tRNA Synthetase Assisted Splicing Mechanisms

亮氨酰-tRNA 合成酶辅助剪接机制

• 批准号: 6735679
• 负责人: SUSAN A MARTINIS
• 金额: $ 21.55万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6735679
• 关键词: RNA; RNA directed DNA polymerase; RNA splicing; Saccharomyces cerevisiae; affinity chromatography; aminoacid tRNA ligase; binding sites; catalyst; enzyme model; fungal genetics; fungal proteins; immunoprecipitation; intermolecular interaction; introns; mitochondria; model design /development; molecular assembly /self assembly; protein structure function; site directed mutagenesis; spliceosomes; yeast two hybrid system

DESCRIPTION (applicant's abstract): The tRNA synthetases are most prominently known for their aminoacylation of tRNA during protein synthesis. However, these enzymes play a number of diverse roles that are also essential to cells. One of these examples is tRNA synthetase-dependent RNA splicing. A novel ternary splicing complex has been identified in yeast where a group I intron called b14 is aided by two proteins, a maturase and a leucyl (Leu)-tRNA synthetase. Tyr-tRNA synthetase (CYT- 18) is the only other tRNA synthetase that has been found to facilitate RNA splicing. However, our preliminary data suggests that the molecular mechanisms by which these two related enzymes promote ribozyme self-splicing activity are quite distinct. Moreover, the Leu-tRNA synthetase-dependent ribozyme is the first example of a two protein: one RNA splicing complex and presents an intriguing elementary model to not only investigate RNA-protein interactions, but also potentially protein-protein interactions which might aid a ribozyme self-splicing reaction. We propose to investigate the Leu-tRNA synthetase-dependent ribozyme splicing reaction and identify discrete determinants that confer this protein synthesis enzyme's unique role in RNA splicing. We have established three-hybrid and RNA-dependent two-hybrid models that show for the first time that the Leu-tRNA synthetase and bI4 maturase can directly, independently, and simultaneously interact with the b14 intron. We have also developed a yeast nucleus-based assay via RT-PCR methods demonstrating that at least one of these protein partners must be bound to faciliate ribozyme splicing activity. We propose to map and identify specific interactions between Leu-tRNA synthetase and the b14 intron that dictate complex assembly and RNA splicing activity. Completion of the proposed specific aims will delineate the undefined splicing role of Leu-tRNA synthetase at the molecular level and provide insight into its inherent determinants that solicited cellular recruitment of this secondary, but also essential activity. Characterization of this simple ternary model offers an important stepping stone in understanding how catalytic ribozymes evolved to more complicated RNP complexes to enhance function and maintain essential biological processes. Moreover, since protein-dependent RNA splicing is critical to the health of human cells as well as to the life cycles of viral, protozoa, and fungal pathogens, elucidation of important molecular determinants required for RNA processing may identify new drug targets for the treatment of infectious disease.

描述（申请人的摘要）：TRNA合成酶是最突出的 以蛋白质合成过程中的tRNA氨基化而闻名。但是，这些 酶扮演着许多对细胞必不可少的角色。之一 这些示例是tRNA合成酶依赖性的RNA剪接。一个新颖的三元 在酵母中已经确定了剪接复合物，其中I组内含子称为B14 通过两种蛋白质，一个成熟酶和亮氨酸（LEU） - tRNA合成酶的帮助。 tyr-tRNA合成酶（Cyt-18）是唯一的其他tRNA合成酶 发现促进RNA剪接。但是，我们的初步数据表明 这两种相关酶促进核酶的分子机制 自我拼写活动是完全不同的。而且，Leu-trna 合成酶依赖性核酶是两种蛋白的第一个例子：一个RNA 剪接复合物，并为不仅为 研究RNA - 蛋白质相互作用，但也可能是蛋白质蛋白 可能有助于核酶自裂反应的相互作用。我们建议 研究Leu-tRNA合成酶依赖性核酶剪接反应和 确定赋予该蛋白质合成酶的离散决定因素 在RNA剪接中的独特作用。我们已经建立了三杂交和RNA依赖性 两个杂交模型首次显示Leu-tRNA合成酶和 BI4成熟酶可以直接，独立并同时与 B14内含子。我们还通过RT-PCR开发了基于酵母核的测定 表明这些蛋白质伴侣中至少有一个必须结合的方法 促进核酶剪接活性。我们建议映射和识别 Leu-tRNA合成酶与B14内含子之间的特定相互作用 决定复杂的组装和RNA剪接活性。拟议的完成 具体目的将描述Leu-tRNA合成酶的未定义剪接作用 在分子水平上，并洞悉其固有的决定因素 征求该次要但基本活性的细胞募集。 这种简单三元模型的表征提供了重要的阶梯 石头了解催化核酶如何演变为更复杂的RNP 复合物来增强功能并维持基本的生物学过程。 此外，由于蛋白质依赖性RNA剪接对于健康至关重要 人类细胞以及病毒，原生动物和真菌的生命周期 病原体，阐明RNA所需的重要分子决定因素 加工可能会确定用于治疗传染性的新药物目标 疾病。