How Does 3' UTR Secondary Structure Program mRNA Transport in Myelination?

3 UTR 二级结构如何控制髓鞘形成中的 mRNA 运输？

基本信息

批准号：
10288149
负责人：
John B Zuchero
金额：
$ 43.3万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10288149
关键词：
3&apos Untranslated Regions Acute Address Antisense Oligonucleotides Autologous Axon Basic Science Binding Biochemical Biology Carrier Proteins Catalogs Cell Differentiation process Cell membrane Cells Chemicals Code Collaborations Communicable Diseases Coupling Data Data Set Demyelinating Diseases Development Disease Explosion Fluorescent in Situ Hybridization Foundations Functional disorder Funding Goals Gold Image In Vitro Knowledge Libraries Maintenance Malignant Neoplasms Mammalian Cell Maps Medicine Messenger RNA Methods Microscopy Modality Molecular Biology Multiple Sclerosis Multiplexed Analysis of Projections by Sequencing Mutagenesis Mutate Mutation Myelin Myelin Basic Proteins Myelin Sheath Natural regeneration Neuraxis Neurobiology Neurons Neurosciences Research Oligodendroglia Patients Pharmaceutical Preparations Prevention strategy Process Production Protocols documentation RNA RNA Sequences RNA Transport RNA vaccine Rattus Regulation Reporter Research Research Project Grants Role Specific qualifier value Spinal Muscular Atrophy Structure Techniques Technology Testing Therapeutic Transcript Translations Variant design dimethyl sulfate experimental study gain of function in vivo insight leukodystrophy mRNA Expression mouse model mutant myelination nervous system disorder new therapeutic target next generation sequencing novel novel therapeutic intervention novel therapeutics oligodendrocyte precursor programs regenerative remyelination single molecule structural biology success targeted treatment therapeutic target transcriptome transcriptome sequencing white matter

项目摘要

PROJECT SUMMARY Recent successes in RNA medicine, including the first-in-class Spinraza treatment for spinal muscular atrophy, demonstrate RNA gain-of-function as a novel therapeutic modality for previously intractable neurological disease and raise the prospect of similar treatments for demyelinating diseases. Such treatments would target oligodendrocytes – the myelinating cells of the central nervous system (CNS) – for a novel and specific strategy for preventative or regenerative RNA medicine. To create myelin, oligodendrocytes extend cell projections that encircle adjacent axons. These concentrically wrapped layers of cell membrane undergo a process called compaction to generate mature myelin. Critical to this process is the localization of a subset of the oligodendrocyte transcriptome to the nascent myelin sheath for local translation. By more than an order of magnitude, the myelin basic protein (MBP) mRNA is the most highly abundant and most highly transported protein-coding transcript in oligodendrocytes, and the drug-induced regulation of its expression, processing, and transport could potentially augment myelination by these cells. Unfortunately, testing the viability of such a strategy is not currently possible due to a lack of understanding of the RNA molecular and structural biology that underlies MBP mRNA transport in oligodendrocytes. In partnership with oligodendrocyte biology collaborators, we have recently begun to address this knowledge gap. We have applied newly invented chemical probing and RNA sequencing technologies to reveal a previously unappreciated repertoire of secondary structures in the MBP 3’ untranslated region (3’ UTR, a region that is known to be necessary for MBP mRNA transport) as well as a catalog of hundreds of other highly transported oligodendrocyte mRNAs. These data suggest features that may be targeted or mimicked with antisense oligonucleotides (ASOs) to modulate MBP mRNA function but need to be rigorously tested. Here, we propose to complete this exploratory research by (1) testing the functional importance of MBP 3’ UTR secondary structures with in-cell mutate-rescue experiments recently invented by our lab and validating these structure-transport relationships through targeted structure perturbation or stabilization facilitated by anti-sense oligonucleotides, and (2) designing a minimal transport-inducing 3’ UTR using insights from high-throughput structure determination and structure-function characterization of all highly transported transcripts in oligodendrocytes. We will evaluate success in both aims through multiple orthogonal methods, including next-generation sequencing, biochemical structure determination, and quantitative single-molecule RNA imaging that we have collaboratively developed for the study of oligodendrocyte projections. The proposed basic science research establishes a previously missing RNA structural biology foundation needed for the design and testing of Spinraza-like ASO therapeutics. This treatment modality could structurally stabilize the 3’ UTRs of myelin-related transcripts to increase their transport and translation in oligodendrocytes, thereby increasing myelin production. Such a drug may be critical in the treatment of otherwise incurable demyelinating diseases.

项目摘要 RNA医学的最新成功，包括用于脊柱肌肉萎缩的第一类Spinraza治疗证明RNA功能获得作为以前棘手的神经系统疾病的新型热方式并提高类似治疗脱髓鞘疾病的前景。这样的治疗将针对少突胶质细胞 - 中枢神经系统（CNS）的髓细胞 - 用于一种新颖而特定的策略用于预防或再生RNA医学。为了创建髓磷脂，少突胶质细胞扩展了细胞项目包围相邻的轴突。这些重复包裹的细胞膜层经历了一个称为的过程压实以产生成熟的髓鞘。这个过程至关重要的是该子集的本地化向新生髓鞘的少突胶质细胞转录组进行局部翻译。超过一个订单大小，髓磷脂碱性蛋白（MBP）mRNA是最丰富且运输最高的mRNA 少突胶质细胞中的蛋白质编码转录本，以及药物诱导的调节其表达，加工和运输可能会通过这些细胞增强髓鞘形成。不幸的是，测试了这样的生存能力由于缺乏对RNA分子和结构生物学的了解，目前不可能进行策略少突胶质细胞中的MBP mRNA转运基础。与少突胶细胞生物学合作者合作，我们最近开始解决这一知识差距。我们应用了新发明的化学探测和 RNA测序技术揭示了先前未批准的二级结构的曲目 MBP 3'未翻译区（3'UTR，一个已知的MBP mRNA传输所必需的区域）作为数百个其他高度运输的少突胶质细胞mRNA的目录。这些数据表明功能可以针对或模仿反义寡核苷酸（ASO）来调节MBP mRNA功能，但需要进行严格测试。在这里，我们建议通过（1）测试功能来完成这项探索性研究 MBP 3’UTR二级结构具有我们最近发明的细胞内突变 - 敏感性实验的重要性实验室并通过靶向结构扰动或稳定来验证这些结构传输关系由反义寡核苷酸促进，（2）使用见解设计最小的传输3'UTR 根据所有高度运输的高通量结构的确定和结构功能表征少突胶质细胞中的转录本。我们将通过多种正交方法评估两个目标的成功，包括下一代测序，生化结构确定和定量单分子 RNA成像我们已经为少突胶质细胞预测而进行了协作开发。提议基础科学研究建立了设计的先前缺失的RNA结构生物学基础和类似脊柱样的ASO治疗的测试。这种治疗方式可以在结构上稳定3'的UTR 髓鞘相关的成绩单以增加少突胶质细胞的运输和翻译，从而增加髓磷脂的产生。这种药物对于治疗原本无法治愈的脱髓鞘疾病可能至关重要。