Leucyl-tRNA Synthetase Assisted Splicing Mechanisms

描述(申请人摘要)：tRNA合成酶是最显著的 以其在蛋白质合成过程中tRNA的氨基酰化而闻名。然而，这些 酶扮演着许多不同的角色，这些角色对细胞也是必不可少的。其中之一 这些例子是依赖tRNA合成酶的RNA剪接。一种新颖的三元组 在酵母中发现了剪接复合体，其中I组内含子称为b14 由两种蛋白质辅助，一个成熟酶和一个亮氨酰(Leu)-tRNA合成酶。 Tyr-tRNA合成酶(Cyt-18)是唯一一种已被 发现可以促进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 自剪接活性是相当不同的。此外，Leu-tRNA 合成酶依赖的核酶是两种蛋白质的第一例：一种RNA 拼接复杂，并提供了一个耐人寻味的基本模型不仅 研究RNA与蛋白质之间的相互作用，以及潜在的蛋白质与蛋白质之间的相互作用 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和 确定赋予这种蛋白质合成酶的离散决定因素 在RNA剪接中的独特作用。我们已经建立了依赖于RNA的三杂交 双杂交模型首次表明Leu-tRNA合成酶和 BI4成熟酶可以直接、独立、同时与 B14内含子。我们还通过RT-PCR开发了一种基于酵母核的检测方法 证明这些蛋白质伴侣中至少有一个必须结合的方法 以促进核酶剪接活性。我们建议绘制和识别 亮氨酸-tRNA合成酶与b14内含子的特异性相互作用 决定复杂的组装和RNA剪接活动。完成拟议的 特定的目标将描述Leu-tRNA合成酶未确定的剪接作用 并提供了对其内在决定因素的洞察 征集蜂窝招聘这一次，也是必不可少的活动。 这个简单的三元模型的特征提供了重要的一步 斯通对催化核酶如何进化为更复杂的RNP的理解 增强功能和维持必要的生物过程的复合体。 此外，由于依赖蛋白质的RNA剪接对人的健康至关重要 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因素 加工可能确定治疗感染性疾病的新药物靶点 疾病。

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.