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

项目摘要 RNA医学领域最近取得的成功，包括针对脊髓性肌萎缩症的一流Spinraza治疗， 证明RNA获得功能作为一种新的治疗方式，以前难治性神经系统疾病 并为脱髓鞘疾病的类似治疗提供了前景。这些治疗将针对 少突胶质细胞-中枢神经系统（CNS）的髓鞘细胞-用于一种新的特异性策略 用于预防或再生RNA药物。为了产生髓鞘，少突胶质细胞延伸细胞突起， 环绕着相邻的轴突这些同心包裹的细胞膜层经历了一个称为 压缩以产生成熟的髓磷脂。这个过程的关键是本地化的子集， 寡突胶质细胞转录组转移到新生髓鞘用于局部翻译。超过了 髓鞘碱性蛋白（MBP）mRNA是最丰富和最高度转运的 蛋白编码转录本，以及药物诱导的其表达、加工和 运输可能潜在地增加这些细胞的髓鞘形成。不幸的是，测试这样一个 由于缺乏对RNA分子和结构生物学的理解， 是少突胶质细胞中MBP mRNA转运的基础。在与少突胶质细胞生物学合作者的合作中， 我们最近开始处理这一知识差距。我们应用了新发明的化学探测技术， RNA测序技术揭示了一个以前未被重视的二级结构库， MBP 3'非翻译区（3' UTR，已知是MBP mRNA转运所必需的区域）也是如此。 数百种其他高转运的少突胶质细胞mRNA的目录。这些数据表明， 可以用反义寡核苷酸（ASO）靶向或模拟以调节MBP mRNA功能，但需要 进行严格的测试。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二级结构的重要性与我们最近发明的细胞内突变拯救实验 通过有针对性结构扰动或稳定化实验和验证这些结构-传输关系 通过反义寡核苷酸促进，和（2）使用洞察设计最小的诱导转运的3' UTR 从高通量结构测定和结构-功能表征的所有高度运输 少突胶质细胞中的转录物。我们将通过多种正交方法评估这两个目标的成功， 包括下一代测序、生化结构测定和定量单分子 RNA成像，我们已经合作开发的研究少突胶质细胞的预测。拟议 基础科学研究建立了设计所需的先前缺失的RNA结构生物学基础 和测试Spinraza-like阿索疗法。这种治疗方式可以在结构上稳定的3'UTR的 髓鞘相关的转录物，以增加它们在少突胶质细胞中的转运和翻译，从而增加 髓鞘生成这种药物可能是治疗其他无法治愈的脱髓鞘疾病的关键。

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.