Insight Into The RNA Processing And Decay Pathways Critical For Proper Neuronal Development And Function Through Focus On Mutations That Cause Pontocerebellar Hypoplasia

货车蹄的原始项目总结/摘要，Ambro R 01 GM 130147 项目摘要 RNA的转录后加工是基因表达的关键调控步骤。许多进化 保守的RNA加工酶介导这些关键的转录后事件。该提案的重点是 与脑桥小脑发育不全（PCH）有关的分子机制，这是一个不断增长的 许多神经系统疾病是由编码RNA加工因子的基因突变引起的。PCH是一个 一组常染色体隐性遗传的神经退行性疾病，其特征在于 小脑和脑桥，往往是致命的第一年内的生活。 导致PCH 1型（PCH 1）的突变发生在编码RNA结构亚基的基因中 外泌体（人Rrp 40、43和45），其在RNA加工和降解中起关键作用。 导致PCH 2、4和5型（PCH 2/4/5）的突变存在于编码tRNA剪接核酸内切酶的基因中 亚基（TSEN 2、15、34和54）。TSEN在tRNA加工中具有充分表征的作用，但也有其他作用。 未定义的函数这些RNase复合物的亚基在进化上都是保守的，并且对于 生存能力PCH 1突变导致主要发生在保守残基中的单个氨基酸取代。 发现这些复合物的多个组分的突变导致PCH强烈表明 RNA加工功能障碍是PCH病理的基础。然而，有限的研究评估了 这些氨基酸取代的功能后果。此外，鉴于常见的疾病病因， RNA外泌体或TSEN复合物中的突变可能会损害常见的RNA靶标或RNA类别 靶点，但受影响的RNA尚未系统地定义。这些与普通生物学的联系 支持我们的工作假设，即导致PCH 1/2/4/5型的突变损害了 一组常见的RNA靶点。我们之前的合作努力提供了模型研究的原理证明 生物体可以深入了解特定的致病氨基酸取代如何损害RNA外泌体 功能在这里，我们利用我们建立的合作和广泛的初步数据来执行一系列 四个目标的机制研究。目的1评估氨基酸变化的功能后果， 发生在PCH使用芽殖酵母;目的2采用小鼠小脑和培养的生化分析 神经元细胞来定义RNA外泌体辅因子，这可能有助于PCH的组织特异性;目的 在芽殖酵母和培养的神经元细胞中进行的3项配对研究，以确定TSEN的共同RNA靶标 和RNA外泌体复合物;最后，Aim 4在果蝇中采用组织特异性RNAi，开始 评估神经元中对特异性RNA外泌体辅因子和TSEN亚基的需求。长期 这项工作的目标是完全确定这些进化上保守的RNA酶复合物的功能， 提供了对可能导致PCH神经功能障碍的分子机制的深入了解。

Original Project Summary/Abstract for van Hoof, Ambro R01 GM130147 PROJECT SUMMARY Post-transcriptional processing of RNA is a critical regulatory step in gene expression. Many evolutionarily conserved RNA processing enzymes mediate these key post-transcriptional events. This proposal focuses on molecular mechanisms linked to PontoCerebellar Hypoplasia (PCH), which serves as a paradigm for a growing number of neurological diseases caused by mutations in genes encoding RNA processing factors. PCH is a group of autosomal recessive neurodegenerative diseases characterized by hypoplasia/atrophy of the cerebellum and pons that is often fatal within the first year of life. Mutations that cause PCH type 1 (PCH1) occur in genes that encode structural subunits of the RNA exosome (human Rrp40, 43, and 45), which plays critical roles in both RNA processing and degradation. Mutations that cause PCH types 2, 4, and 5 (PCH2/4/5) lie in genes that encode tRNA splicing endonuclease subunits (TSEN2, 15, 34, and 54). TSEN has a well-characterized role in tRNA processing but also other yet undefined functions. The subunits of these RNase complexes are all evolutionarily conserved and essential for viability. PCH1 mutations cause single amino acid substitutions that primarily occur in conserved residues. The discovery that mutations in multiple components of these complexes cause PCH strongly suggests that RNA processing dysfunction underlies PCH pathology. However, limited studies have assessed the functional consequences of these amino acid substitutions. Furthermore, given the common disease etiology, mutations in either the RNA exosome or TSEN complex could impair common RNA targets or classes of RNA targets, but the RNAs affected have not been systematically defined. These links to common biology strongly support our working hypothesis that mutations that cause PCH Types 1/2/4/5 impair the processing of a common set of RNA targets. Our previous collaborative efforts provide proof of principle that studies in model organisms can provide insight into how specific disease-causing amino acid substitutions impair RNA exosome function. Here we draw on our established collaboration and extensive preliminary data to perform a series of mechanistic studies in four aims. Aim 1 assesses the functional consequences of amino acid changes that occur in PCH using budding yeast; Aim 2 employs biochemical analysis in mouse cerebellum and cultured neuronal cells to define RNA exosome cofactors that could contribute to the tissue-specific nature of PCH; Aim 3 couples studies in budding yeast and cultured neuronal cells to identify common RNA targets of the TSEN and RNA exosome complexes; and, finally, Aim 4 employs tissue-specific RNAi in Drosophila to begin to assess the requirement for specific RNA exosome cofactors and TSEN subunits in neurons. The long-term goal of this work is to fully define the function of these evolutionarily conserved RNase complexes while providing insight into molecular mechanisms that could contribute to neurological dysfunction in PCH.