URIL tags for intracellular RNA tracking and RNP proximity labeling

项目摘要/摘要 如果在基因编码RNA的内部位置选择性地安装修复体基团的简便方法是 那么就有可能对天然的RNA和核糖核蛋白复合体(RNPs)进行修饰 结构和细胞内环境，从而提升了对RNA折叠、运输、寿命、相互作用和 调控途径。该应用程序的目标是测试紧凑的富铀内部循环的程度 (URIL)位点可作为结构RNA中的通用靶向基序。如果有可能有选择性地 用化学探针定位uril基序，然后将uril位点与蛋白质结合RNA基序并列 将能够对核糖核蛋白复合体(RNPs)进行追踪和化学修饰。这将使 用尿素的荧光和邻近(生物素)标记阐明基序特异的RNA定位和相互作用组 RNPs；这种以基序为中心的无偏见的交互作用组读出用现有的方法是不可能的。我们 假设适当修饰的、尿素靶向双面肽核酸(BPNAs)可以使 胞内荧光脲(FLURIL)RNA跟踪和邻近标记尿素(PLURIL)RNPs， 分别进行了分析。我们的计划首先是合成bPNA探针，然后是严格的体外和 与现有工具和已知的互动组合作伙伴进行细胞内评估、优化和验证。FLURIL RNP标记将以MS2标记为基准，这是RNA跟踪的黄金标准。PLURIL标记 将通过其在识别已知RNPs方面的有效性进行测试。此外，我们将测试uril标记的程度。 可用于探索肌萎缩侧索硬化症细胞内相关RNP生物学 (ALS)，使用患者来源的细胞。肌萎缩侧索硬化症的病理研究是一个高度活跃的领域，也是人们关注的焦点。 有两种主要形式：C9-ALS和FUS连锁ALS。虽然C9-ALS代表了大多数ALS病例， 与FUS相关的肌萎缩侧索硬化症最常见于青少年、侵袭性早发病例；值得注意的是，病理上 这两种形式的机制似乎是截然不同的。以C9orf72 RNA为核心的RNP失调生物学 (C9-ALS)和U1SnRNA(FUS连锁ALS)鉴定这些转录本是uril标签探针的主要底物。 以往研究的严谨性在于支持细胞内的实质性初步和已发表的数据 URIL-RNPs的荧光标记和邻近标记。这些数据构成了一个强有力的科学前提 Motif特异性uril标记作为一种广泛使能的发现工具在ALS病理中的独特应用 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.