URIL tags for intracellular RNA tracking and RNP proximity labeling

项目总结/摘要 如果有一种简单的方法，在遗传编码RNA的内部位点上选择性地安装辅基， 可用，那么就有可能在天然的细胞中修饰RNA和核糖核蛋白复合物（RNP）。 结构和细胞内环境，从而提升了对RNA折叠，运输，寿命，相互作用组和 调控途径。本申请的目的是测试紧凑型富U内部回路 URIL位点可以用作结构化RNA中的一般靶向基序。如果有可能选择性地 用化学探针靶向URIL基序，然后将URIL位点与蛋白结合RNA基序并置 将能够跟踪和化学修饰核糖核蛋白复合物（RNP）。这将使 通过URIL的荧光和邻近（生物素）标记阐明基序特异性RNA定位和相互作用组 RNP;这种无偏的以基序为中心的相互作用组读出用现有方法是不可能的。我们 假设适当修饰的、靶向URIL的双面肽核酸（bPNAs）能够 胞内荧光URIL（FLURIL）RNA追踪和URIL（PLURIL）RNP的邻近标记， 分别我们提出的计划从bPNA探针的合成开始，然后进行严格的体外和 使用现有工具和已知的相互作用物组伙伴进行细胞内评估、优化和验证。氟脲嘧啶 RNP标记将以MS 2标记为基准，MS 2标记是RNA追踪的金标准。PLURIL标记 将通过其在识别已知RNP方面的有效性进行测试。此外，我们将测试URIL标记 可用于在肌萎缩侧索硬化的细胞内环境中探测疾病相关的RNP生物学 (ALS)使用患者来源的细胞。ALS病理学的研究是一个高度活跃的领域， 两种主要形式：C9-ALS和Fus-linked ALS。虽然C9-ALS代表了大多数ALS病例， Fus-linked ALS最常见于青少年、攻击性早发性病例;值得注意的是，病理性ALS患者的年龄在10岁以下。 这两种形式的机制似乎是不同的。以C9 orf 72 RNA为中心的RNP生物学失调 （C9-ALS）和U1 snRNA（Fus-linked ALS）鉴定这些转录物为URIL标签探针的主要底物。 先前研究的严谨性在于大量的初步和发表的数据支持细胞内 URIL-RNP的荧光和邻近标记。这些数据构成了一个强有力的科学前提， 以及基序特异性URIL标记作为ALS病理学中广泛启用的发现工具的独特应用 以及其他以RNP为中心的疾病。

PROJECT SUMMARY / ABSTRACT If a facile method to site-selectively install prosthetic groups at internal sites in genetically-encoded RNA were available, then it would be possible to modify RNAs and ribonucleoprotein complexes (RNPs) in native structural and intracellular context, thus elevating studies on RNA folding, trafficking, lifetime, interactomes and regulatory pathways. The objective of this application is to test the extent to which compact U-rich internal loop (URIL) sites can be used as a general targeting motif in structured RNAs. If it were possible to selectively target the URIL motif with chemical probes, then juxtaposition of the URIL site with protein binding RNA motifs would enable tracking and chemical modification of ribonucleoprotein complexes (RNPs). This would enable elucidation of motif-specific RNA location and interactome by fluorogenic and proximity (biotin) labeling of URIL RNPs; such unbiased motif-centered interactome readout is not possible with existing methods. We hypothesize that appropriately modified, URIL-targeting bifacial peptide nucleic acids (bPNAs) could enable intracellular fluorogenic URIL (FLURIL) RNA tracking and proximity labeling of URIL (PLURIL) RNPs, respectively. Our proposed plan begins with the synthesis of bPNA probes, followed by rigorous in vitro and intracellular evaluation, optimization and validation with existing tools and known interactome partners. FLURIL RNP tagging will be benchmarked against MS2-labeling, the gold standard in RNA tracking. PLURIL tagging will be tested by its efficacy in identification of known RNPs. Further, we will test the extent to which URIL tags can be used to probe disease-relevant RNP biology in the intracellular context of amyotrophic lateral sclerosis (ALS), using patient-derived cells. Investigation of ALS pathology is a highly active area, with attention focused on two major forms: C9-ALS and Fus-linked ALS. While C9-ALS represents a majority of ALS cases, Fus-linked ALS is most commonly found in juvenile, aggressive early-onset cases; notably, the pathological mechanisms of these two forms appear to be distinct. Dysregulated RNP biology centered on C9orf72 RNA (C9-ALS) and U1snRNA (Fus-linked ALS) identifies these transcripts as prime substrates for URIL tag probes. The rigor in the prior research lies in the substantive preliminary and published data supporting intracellular fluorogenic and proximity labeling of URIL-RNPs. These data form a strong scientific premise for the impactful and unique application of motif-specific URIL-tagging as a broadly enabling discovery tool in ALS pathology and other RNP-centered diseases.