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: 10388656
• 负责人: John Conor Moran
• 金额: $ 5.18万
• 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/10388656
• 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; Genetic Transcription; Genetic Translation; 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; Multiplexed Analysis of Projections by Sequencing; 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; 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; Transact; Transcript; Transcription Process; Transfer RNA; Translations; Variant; base; cell type; crosslink; cytochrome c oxidase; dimethyl sulfate; genetic variant; innovation; interest; mRNA Expression; mRNA Stability; mitochondrial messenger RNA; mouse model; mutant; oligomycin sensitivity-conferring protein; polypeptide; prevent; reconstitution; ribosome profiling; 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，会导致儿童不可逆转的发育退化。 目前，尚无可用的治疗方案，大多数儿童都活不到两岁生日。 LS 是 生化特征是由于多种可能突变导致的线粒体生物能缺陷 线粒体蛋白，可以在线粒体和核 DNA 中编码。在这里，我们将重点关注 两种 LS 蛋白：富含亮氨酸的五肽重复基序含有蛋白 (LRPPRC) 和 考西（TACO1）。 LRPPRC 的丢失会降低所有 mRNA 的稳定性，并导致 mRNA 丢失 多腺苷酸化和线粒体翻译异常的出现。 TACO1 的丢失特别可以防止 COX1 mRNA 的翻译。尽管有了这些知识，但所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 调控的多种机制不可或缺的一部分 的基因表达。通过结构探测应用进行 RNA 结构分析的最新进展 化学品与高通量测序（如DMS-MaPseq）相结合开辟了一个新领域 极大地扩展了可用RNA结构信息的数量。有了这项技术，这里 我们建议检验 LRPPRC 或 TACO1 突变导致下游缺陷的假设 线粒体 mRNA 的折叠以及随后异常的 mtDNA 基因表达。测试 在这个假设中，我们将遵循两个目标，并对每个基因使用人类细胞系敲除（KO），我们 是通过使用创新的 CRISPR-Cas9 基因编辑方法生成的。在目标 1 中，KO 线将是 用野生型或疾病突变基因变体重建，并根据细胞特征进行生理学表征 线粒体生物能学、线粒体 RNA 加工和稳定性，以及线粒体翻译。 现有 LRPPRC-KO 细胞系的初步数据已揭示线粒体表型 与 LS 患者的情况相符。随后将建立这些细胞内 mRNA 结合位点 使用 PAR-CLIP 方法分析野生型细胞中的蛋白质。最后，我们将利用核糖体分析测定 确定 LRPPRC 和 TACO1 在翻译过程中是否结合其目标 mRNA。在目标 2 中我们将使用 DMS-MaPseq 用于探测 KO 中整个线粒体转录组的天然结构并进行重组 细胞系。为了确保这项事业取得成功，我们将利用与 Silvia Rouskin 博士的合作， DMS-MaPseq 的先驱。我们期望回答有关这些蛋白质如何相互作用的基本问题 mRNA，影响它们的折叠，并最终影响它们的稳定性、修饰和翻译。为了充分了解 几种 LS 形式背后的基本致病机制对于 NICHD 的使命至关重要，并且 开发针对此类疾病的治疗方法的基本先决条件。从这个角度来看， 拟议的项目是针对我成为一名熟练的医师科学家的培训而量身定制的。