Structural and Functional Characterization of Pontocerebellar Hypoplasia Associated Nucleases

项目摘要/摘要 RNA加工是一个基本的细胞过程，当失调时，它是发展的基础 神经系统疾病。含有核酸酶的复合体的几个突变导致桥小脑发育不良 (PCH)，一种严重的神经疾病，通常会导致产前死亡。大多数PCH病例与 TRNA剪接内切酶(Tsen)复合体的突变，该复合体负责tRNA的切割 TRNA成熟前的内含子及其附属蛋白CLP1，它是tRNA的关键负调控因子 拼接。酵母中任何单一Tsen蛋白的基因缺失，被改造为含有不含内含子的tRNAs， 显示出是致命的，这表明Tsen复合体可能具有tRNA以外的底物，这可能 是五氯环己烷发展的基础。同样，另一种核酸酶的突变，Egr 1(TOE1)的靶标，a 死烯基酶和3‘-外切核酸酶也与PCH有关。TOE1是唯一一种被认为成熟的死烯基酶 但它也兼职于其他细胞途径，突显出对其有多少了解 在蛋白质复合体中的作用。 要确定TOE1、CLP1和Tsen蛋白突变是如何导致PCH的，仍然有一个关键的 需要了解这些复合体如何组装、识别和处理RNA，以及它们的酶是如何 活动是受监管的。确定这些蛋白质在细胞中的健康作用对于确定它们的 功能障碍会导致PCH。我们的目标是通过以下拟议目标解决这些关键问题。在……里面 目标1，将使用结构和分子技术来确定Tsen复合体如何识别和 加工tRNA和其他RNA底物。在目标2中，我们将确定CLP1/Tsen复合体是如何 在分子和细胞水平上受到调控，以及PCH突变如何破坏它们的调控。此外，在目标3中， 我们将利用蛋白质组学和分子生物学来确定PCH突变如何改变TOE1的功能和调节 接近了。 拟议的工作具有重要意义，因为它将提供对如何了解PCH的结构描述 突变可能会干扰一系列PCH连接蛋白的复合体的形成、稳定性或功能。这 这项工作将进一步深入了解这些蛋白质复合体导致PCH的共同机制。 此外，这里的工作将表征这些核酸酶的新的RNA处理角色。

PROJECT SUMMARY/ABSTRACT RNA processing is an essential cellular process that when dysregulated underlies the development of neurological diseases. Several mutations in nuclease containing complexes cause pontocerebellar hypoplasia (PCH), a severe neurological disorder that often leads to prenatal death. Most cases of PCH are linked to mutations in the tRNA Splicing Endonuclease (TSEN) Complex, which is responsible for the cleavage of tRNA introns prior to tRNA maturation, and its accessory protein, CLP1, which is a critical negative regulator of tRNA splicing. Genetic deletion of any single TSEN protein in yeast, engineered to have tRNAs without introns, was shown to be lethal, suggesting that the TSEN complex likely has substrates beyond the tRNAs, which may underlie the development of PCH. Likewise, mutations in another nuclease, target of Egr 1 (TOE1), a deadenylase and 3’-exonuclase, are also are linked to PCH. TOE1 is the only deadenylase believed to mature snRNAs, but it also moonlights in other cellular pathways, highlighting how much is yet to understand about its role in protein complexes. To determine how mutations in TOE1, CLP1, and TSEN proteins lead to PCH, there remains a critical need to understand how these complexes assemble, recognize and process RNAs, and how their enzymatic activity is regulated. Characterizing healthy cellular roles of these proteins is essential to determining how their dysfunction causes PCH. We aim to address these critical questions through the following proposed Aims. In Aim 1, Structural and molecular techniques will be used to identify how the TSEN complex recognizes and processes tRNAs and other RNA substrates. In Aim 2, we will determine how the CLP1/TSEN complex are regulated at the molecular and cellular level and how PCH mutations disrupt their regulation. Further, in Aim 3, we will identify how PCH mutations alter TOE1 function and regulation, using proteomics and molecular biology approaches. The proposed work is significant because it will provide a structural description for how known PCH mutations may interfere with complex formation, stability, or function for a range of PCH-linked proteins. This work will further provide insight into shared mechanisms by which these protein complexes cause PCH. Furthermore, the work here will characterize new RNA processing roles for these nucleases.