Mitochondrial mRNA structure as a driver of Leigh Syndrome

Leigh综合征(LS)是一种导致儿童不可逆转的发育退化的线粒体疾病。 目前，还没有可用的治疗方案，而且大多数儿童都活不到两岁生日。LS是 生物化学的特征是由于各种可能的突变而导致的线粒体生物能有缺陷 线粒体蛋白质，可在线粒体和核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包含蛋白(LRPPRC)和反式激活因子 Coxi(TACO1)。LRPPRC的丢失降低了所有mRNAs的稳定性，也导致了mRNAs的丢失 多聚腺苷酸化和线粒体异常翻译的出现。TACO1的损失特别防止 COX1基因的翻译。尽管有这些知识，但其中涉及的分子机制仍不清楚。 RNA折叠成复杂的结构，这些结构是RNA调控的不同机制所不可或缺的 关于基因表达的。结构探测技术在RNA结构分析中的应用研究进展 化学品与高通量测序(如DMS-MaPseq)相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mRNAs的折叠和随后mtDNA基因的异常表达。为了测试 在这个假设中，我们将遵循两个目标，并将对每个基因使用人类细胞系基因敲除(KO)，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线将是 用野生型或疾病突变基因变异体重组的，在细胞方面具有生理特征的 以及线粒体生物能量学、线粒体RNA的加工和稳定性以及线粒体的翻译。 已有的LRPPRC-KO细胞系的初步数据已告知线粒体表型 与LS患者相容。随后将建立这些基因的细胞内mRNA结合位点 用PAR-CLIP方法研究野生型细胞中的蛋白质。最后，我们将利用核糖体图谱分析来 确定LRPPRC和TACO1是否在翻译过程中结合其目标mRNAs。在《目标2》中，我们将使用 DMS-MaPseq在KO和重组的线粒体转录组中的天然结构的探测 细胞系。为了确保这一事业的成功，我们将利用我们与西尔维娅·卢斯金博士的合作， DMS的先驱--MaPseq。我们希望回答有关这些蛋白质如何相互作用的基本问题。 影响它们的折叠，最终影响它们的稳定性、修饰和翻译。要充分了解 几种LS类型的基本致病机制对NICHD任务和 开发针对这种疾病的治疗方法是基本的先决条件。从这个角度来看， 提议的项目是为我成为一名熟练的内科科学家而量身定做的。

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.