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Structural and Functional Characterization of Pontocerebellar Hypoplasia Associated Nucleases

桥小脑发育不全相关核酸酶的结构和功能表征

基本信息

批准号：
10734463
负责人：
Cassandra K Hayne
金额：
$ 24.9万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-12 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10734463
关键词：
Address Binding Biochemical Biological Biological Assay Biology Cell physiology Cells Cessation of life Complement Complex Cryoelectron Microscopy Defect Development Disease Engineering Exonuclease Family Fluorescence Microscopy Functional disorder Genetic Goals Health Human Hydroxyl Radical In Vitro Introns Lead Left Link Mass Spectrum Analysis Microscopy Molecular Molecular Biology Mutation Neurodegenerative Disorders Neurodevelopmental Disorder Nuclear Pathway interactions Polynucleotide 5&apos -Hydroxyl-Kinase Pontocerebellar hypoplasia Process Production Proteins Proteomics Protocols documentation RNA RNA Binding RNA Processing RNA Splicing Regulation Research Personnel Role Small Nuclear RNA Substrate Specificity Surface Techniques Therapeutic Transfer RNA Untranslated RNA Validation Work Yeasts biological adaptation to stress cell growth regulation circular RNA crosslink endonuclease human disease insight link protein mutant nervous system disorder nuclease prenatal prevent protein complex protein function protein protein interaction reconstitution transcriptome sequencing

项目摘要

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.

项目总结/文摘