Leucyl-tRNA Synthetase Assisted Splicing Mechanisms

亮氨酰-tRNA 合成酶辅助剪接机制

• 批准号: 6320999
• 负责人: SUSAN A MARTINIS
• 金额: $ 23.65万
• 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 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6320999
• 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（申请人摘要）：tRNA合成酶是最主要的 以其在蛋白质合成过程中tRNA的氨酰化而闻名。但这些 酶起着许多对细胞也是必不可少的不同作用。之一 这些例子是tRNA合成酶依赖的RNA剪接。一种新型三元 已经在酵母中鉴定出剪接复合体，其中称为b14的I组内含子 由两种蛋白质辅助，成熟酶和亮氨酰（Leu）-tRNA合成酶。 Tyr-tRNA合成酶（CYT- 18）是唯一的其他tRNA合成酶， 有助于RNA剪接。然而，我们的初步数据表明， 这两种相关酶促进核酶的分子机制 自我剪接活性是相当不同的。此外，Leu-tRNA 合成酶依赖性核酶是第一个两种蛋白质：一种RNA 拼接复杂，并提出了一个有趣的基本模型，不仅 研究RNA与蛋白质的相互作用，也可能研究蛋白质与蛋白质的相互作用 可能有助于核酶自剪接反应的相互作用。我们建议 研究Leu-tRNA合成酶依赖的核酶剪接反应， 鉴定赋予这种蛋白质合成酶 在RNA剪接中的独特作用。我们已经建立了三杂交和RNA依赖 双杂交模型首次表明Leu-tRNA合成酶和 bI 4成熟酶可以直接、独立和同时与 b14内含子。我们还通过RT-PCR开发了一种基于酵母细胞核的检测方法 证明这些蛋白质伴侣中的至少一种必须结合 以促进核酶剪接活性。我们建议绘制并识别 Leu-tRNA合成酶和b14内含子之间的特异性相互作用， 决定了复杂的装配和RNA剪接活性。完成建议 具体的目标将描述Leu-tRNA合成酶的未定义的剪接作用 在分子水平，并提供洞察其内在的决定因素， 这是一种次要的，但也是必不可少的活动。 这个简单的三元模型的特征提供了一个重要的步骤 理解催化核酶如何进化为更复杂的RNP的关键 复合物，以增强功能和维持基本的生物过程。 此外，由于蛋白质依赖的RNA剪接对人类的健康至关重要， 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簇 处理可以识别用于治疗感染性疾病的新药靶点， 疾病