Mitochondrial mRNA structure as a driver of Leigh Syndrome

线粒体 mRNA 结构作为 Leigh 综合征的驱动因素

• 批准号: 10545718
• 负责人: John Conor Moran
• 金额: $ 5.27万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10545718
• 关键词: ATP Synthesis Pathway; Aerobic; Affect; Age; Appearance; Attenuated; Binding; Binding Sites; Biochemical; Bioenergetics; Biogenesis; Biological Assay; CRISPR/Cas technology; Cell Line; Chemicals; Child; Childhood; Collaborations; Complex; Cultured Cells; Cytochrome-c Oxidase Deficiency; Cytoplasmic Granules; DNA; Data; Defect; Development; Developmental Delay Disorders; Disease; Ensure; Enzymes; Etiology; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Goals; High-Throughput Nucleotide Sequencing; Human; Human Cell Line; Immunoprecipitation; Infant; Knock-out; Knowledge; Lead; Leigh Disease; Lesion; Leucine; Medicine; Messenger RNA; Mission; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Proteins; Mitochondrial RNA; Modification; Molecular; Mutate; Mutation; National Institute of Child Health and Human Development; Nerve Degeneration; Newborn Infant; Nuclear; Oxidative Phosphorylation; PTGS1 gene; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Phenotype; Physicians; Physiological; Poly A; Polyadenylation; Polyadenylation Pathway; Property; Protein Biosynthesis; Proteins; RNA; RNA Folding; RNA Processing; RNA Stability; RNA metabolism; Regulation; Respiratory Chain; Ribonucleosides; Ribosomal RNA; Role; Scientist; Structural Models; Structural defect; Structure; System; Technology; Testing; Therapeutic; Training; Trans-Activators; Transcript; Transcription Process; Transfer RNA; Translations; Variant; base; cell type; complex IV; crosslink; cytochrome c oxidase; dimethyl sulfate; genetic variant; innovation; interest; mRNA Expression; mRNA Stability; mRNA Translation; mitochondrial messenger RNA; mouse model; mutant; oligomycin sensitivity-conferring protein; polyadenylated messenger RNA; polypeptide; posttranscriptional; prevent; reconstitution; ribosome profiling; skills; success; therapeutic target; transcriptome

Leigh syndrome (LS) is a mitochondrial disease that causes irreversible developmental regression in children. Currently, no treatment options are available, and most children do not live to see their second birthday. LS is biochemically characterized by defective mitochondrial bioenergetics due to a variety of possibly mutated mitochondrial proteins, which can be encoded in both mitochondrial and nuclear DNA. Here, we will focus on two LS proteins: leucine rich pentacotripeptide repeat motif containing protein (LRPPRC), and transactivator of COXI (TACO1). Loss of LRPPRC decreases stability of all mRNAs, and also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. Loss of TACO1 specifically prevents translation of COX1 mRNA. Despite this knowledge, the molecular mechanisms involved remain unknown. RNAs fold into complex structures that are integral to the diverse mechanisms underlying RNA regulation of gene expression. Recent development of RNA structure profiling through the application of structure-probing chemicals combined with high-throughput sequencing (such as DMS-MaPseq) has opened a new field that enormously expands the amount of RNA structural information available. With this technology available, here we propose to test the hypothesis that mutations in either LRPPRC or TACO1 lead to a downstream defect in the folding of mitochondrial mRNAs and subsequently to aberrant mtDNA gene expression. To test this hypothesis, we will follow two Aims and will use human cell lines knockout (KO) for each gene, which we are generating by using innovative CRISPR-Cas9 gene-editing approaches. In Aim 1, the KO lines will be reconstituted with wild-type or disease mutant gene variants and characterized physiologically in terms of cellular and mitochondrial bioenergetics, mitochondrial RNA processing and stability, as well as mitochondrial translation. Preliminary data on already available LRPPRC-KO cell lines have informed mitochondrial phenotypes compatible with those of LS patients. This will follow with establishment of in-cello mRNA binding sites of these proteins in wild-type cells using PAR-CLIP approaches. Finally, we will utilize ribosome-profiling assays to determine whether LRPPRC and TACO1 are binding their target mRNAs during translation. In Aim 2 we will use DMS-MaPseq to probe the native structures of the entire mitochondrial transcriptome in the KO and reconstituted cell lines. To ensure success in this undertaking, we will capitalize on our collaboration with Dr. Silvia Rouskin, the pioneer of DMS-MaPseq. We expect to answer basic questions regarding how these proteins interact with mRNA, affect their folding, and ultimately their stability, modification and translation. To fully understand the basic pathogenic mechanisms underlying the several LS forms is critical to the NICHD mission and a fundamental pre-requisite to develop therapeutics to target this kind of disorders. With this perspective, the proposed project is tailored to my training towards becoming a skilled physician scientist.

Leigh综合征（LS）是一种线粒体疾病，可导致儿童不可逆的发育退化。 目前，没有治疗方案，大多数儿童活不到第二个生日。LS是 生化特征为由于多种可能的突变导致线粒体生物能量学缺陷 线粒体蛋白质，其可以在线粒体和核DNA中编码。在这里，我们将重点介绍 两种LS蛋白：富含亮氨酸的五肽重复基序蛋白（LRPPRC）和 COXI（TACO 1）。LRPPRC的缺失降低了所有mRNA的稳定性，也导致mRNA的缺失。 多聚腺苷酸化和异常线粒体翻译的出现。TACO 1的缺失可以防止 COX 1 mRNA的翻译。尽管有这些知识，但涉及的分子机制仍然未知。 RNA折叠成复杂的结构，这些结构是RNA调节的各种机制的组成部分 的基因表达。结构探针技术在RNA结构分析中的应用进展 化学品与高通量测序（如DMS-MaPseq）的结合开辟了一个新的领域， 极大地扩展了可用的RNA结构信息的数量。有了这项技术， 我们建议检验LRPPRC或TACO 1突变导致下游缺陷的假设， 在线粒体mRNA的折叠和随后的异常mtDNA基因表达。测试 根据这一假设，我们将遵循两个目标，并将使用人细胞系敲除（KO）每个基因，我们 是通过使用创新的CRISPR-Cas9基因编辑方法产生的。在目标1中，KO线将 用野生型或疾病突变基因变体重构，并在细胞生物学方面进行生理学表征， 以及线粒体生物能量学、线粒体RNA加工和稳定性以及线粒体翻译。 关于已经可用的LRPPRC-KO细胞系的初步数据已经告知了线粒体表型 与LS患者一致。这将遵循这些蛋白的细胞内mRNA结合位点的建立。 使用PAR-CLIP方法在野生型细胞中表达蛋白。最后，我们将利用核糖体分析法， 确定LRPPRC和TACO 1是否在翻译过程中结合其靶mRNA。在目标2中，我们将使用 DMS-MaPseq以探测KO中整个线粒体转录组的天然结构，并重构 细胞系为了确保这项工作取得成功，我们将利用与西尔维娅·罗金博士的合作， DMS-MaPseq的先驱。我们希望能够回答关于这些蛋白质如何与 mRNA影响它们的折叠，并最终影响它们的稳定性、修饰和翻译。全面了解 几种LS形式的基本致病机制对于NICHD的使命至关重要， 这是开发针对这类疾病的治疗方法的基本先决条件。从这个角度来看， 建议的项目是针对我的训练，成为一个熟练的医生科学家。