Insight into the RNA processing and decay pathways critical for proper neuronal development and function through focus on mutations that cause Pontocerebellar Hypoplasia

通过关注导致脑桥小脑发育不全的突变，深入了解对神经元正常发育和功能至关重要的 RNA 加工和衰变途径

• 批准号: 10414387
• 负责人: ANITA H. CORBETT
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-11 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10414387
• 关键词: Affect; Amino Acid Substitution; Amino Acids; Animal Model; Atrophic; Behavior; Biochemical; Biological Models; Biology; Brain region; Cells; Cerebellum; Chemistry; Child; Cleaved cell; Collaborations; Complex; Couples; Critical Pathways; Data; Defect; Degradation Pathway; Disease; Drosophila genus; Enzymes; Etiology; Eukaryota; Event; Functional disorder; Gene Expression; Genes; Genetic Transcription; Goals; Human; Immunoprecipitation; Impairment; Learning; Life; Link; Longevity; Mass Spectrum Analysis; Mediating; Memory; Messenger RNA; Molecular; Mus; Mutation; Nature; Neurodegenerative Disorders; Neurologic Dysfunctions; Neurons; Pathology; Pathway interactions; Play; Pontine structure; Pontocerebellar hypoplasia; Post-Transcriptional RNA Processing; Proteins; Publishing; RNA; RNA Decay; RNA Degradation; RNA Interference; RNA Processing; RNA Splicing; RNA analysis; Research Design; Ribonucleases; Role; Saccharomycetales; Series; Specificity; Structure; Substrate Specificity; Testing; Tissues; Transcript; Transfer RNA; Work; Yeast Model System; Yeasts; brain morphology; cell growth; cofactor; disease-causing mutation; endonuclease; exosome; experimental study; fly; human disease; in vivo; insight; molecular pathology; mutant; nervous system disorder; neuron development; public health relevance; transcriptome sequencing

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.

项目摘要 RNA的转录后处理是基因表达的关键调节步骤。许多进化 保守的RNA加工酶介导了这些关键的转录后事件。该提议重点 与Pontocerebellar发育不全（PCH）相关的分子机制，它是增长的范式 由编码RNA处理因子的基因突变引起的神经疾病数量。 PCH是一个 常染色体隐性神经退行性疾病，这些疾病为特征于发育不全/萎缩的特征 小脑和庞斯在生命的第一年中通常是致命的。 引起PCH 1型（PCH1）的突变发生在编码RNA结构亚基的基因中 外泌体（人RRP40、43和45），在RNA加工和降解中起关键作用。 引起PCH类型2、4和5（PCH2/4/5）的突变在于编码tRNA剪接核酸内切酶的基因 亚基（TSEN2、15、34和54）。 TSEN在tRNA处理中具有良好的作用，但其他 未定义的功能。这些RNase复合物的亚基都是进化保守的，对于 生存能力。 PCH1突变导致主要发生在保守残基中的单氨基酸取代。 这些复合物多种组分中的突变引起PCH的发现强烈表明 RNA处理功能障碍是PCH病理学的基础。但是，有限的研究评估了 这些氨基酸取代的功能后果。此外，鉴于常见的病因学， 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 目标，但是受影响的RNA尚未系统地定义。这些与共同生物学的链接强烈 支持我们的工作假设，即导致PCH类型1/2/4/5的突变损害了处理 共同的RNA靶标。我们以前的合作努力提供了原则的证明，表明模型中的研究 生物体可以洞悉特定引起疾病的氨基酸替代如何损害RNA外泌体 功能。在这里，我们借鉴了我们既定的合作和广泛的初步数据，以执行一系列 四个目标的机械研究。 AIM 1评估氨基酸变化的功能后果 使用芽酵母在PCH中发生； AIM 2在小脑小脑中采用生化分析和培养 神经元细胞定义RNA外泌体辅助因子，可能有助于PCH的组织特异性。目的 3对夫妻在萌芽的酵母和培养的神经元细胞中进行研究，以鉴定TSEN的常见RNA靶标 和RNA外泌体配合物；最后，AIG 4在果蝇中采用组织特异性RNAi开始 评估神经元中特定RNA外泌体辅因子和TSEN亚基的需求。长期 这项工作的目标是充分定义这些进化保守的RNase复合物的功能，而 提供有关可能导致PCH神经功能障碍的分子机制的见解。

项目成果

Yeast Dxo1 is required for 25S rRNA maturation and acts as a transcriptome-wide distributive exonuclease.

• DOI: 10.1261/rna.078952.121
• 发表时间: 2022-05
• 期刊: RNA (New York, N.Y.)

Modeling Pathogenic Variants in the RNA Exosome.

• DOI: pii: e1166
• 发表时间: 2020
• 期刊: RNA & disease (Houston, Tex.)
• 作者: de Amorim J;Slavotinek A;Fasken MB;Corbett AH;Morton DJ
• 通讯作者: Morton DJ

An endogenous PI3K interactome promoting astrocyte-mediated neuroprotection identifies a novel association with RNA-binding protein ZC3H14.

• DOI: 10.1074/jbc.ra120.015389
• 发表时间: 2021-01
• 期刊: The Journal of biological chemistry
• 作者: Alqawlaq S;Livne-Bar I;Williams D;D'Ercole J;Leung SW;Chan D;Tuccitto A;Datti A;Wrana JL;Corbett AH;Schmitt-Ulms G;Sivak JM
• 通讯作者: Sivak JM

Dissecting the roles of Cse1 and Nup2 in classical NLS-cargo release in vivo.

• DOI: 10.1111/tra.12759
• 发表时间: 2020-10
• 期刊: Traffic (Copenhagen, Denmark)
• 作者: Lange A;Fasken MB;Stewart M;Corbett AH
• 通讯作者: Corbett AH

Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway

• DOI: 10.1073/pnas.2020429118
• 发表时间: 2021-03-09
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Hurtig, Jennifer E.;Steiger, Michelle A.;van Hoof, Ambro
• 通讯作者: van Hoof, Ambro