Integrating RNAs into signaling pathways by engineering covalent RNA modification

描述（由申请人提供）：对调控网络的分子水平理解推动了生物医学的进步，但主要集中在蛋白质网络上。非编码RNA（ncRNA），包括大的ncRNA（lncRNA），也发挥重要作用。例如，Xist lncRNA的缺失导致小鼠的癌症。然而，lncRNA的功能范围，其作用机制及其对治疗重要途径的影响在很大程度上仍然未知。鉴定高化学计量或稳定的RNA-蛋白质相互作用的方法为RNA提供了有力的见解，但错过了重要的相互作用，例如RNA解旋酶与其底物的相互作用，这些相互作用是短暂的或低化学计量的。类似地，低丰度表达的lncRNA对调控很重要，但与它们相互作用的蛋白质在很大程度上是未知的。为了满足这些需求，我们正在开发一个平台来设计共价RNA修饰，从而产生新的方法。首先，共价RNA标签的开发将有助于发现与低丰度lncRNA相互作用的蛋白质。第二，这个相同的平台被用于开发共价追踪策略，其中感兴趣的蛋白质被工程化以在瞬时结合的RNA上留下共价标记。这种共价修饰将用作纯化手柄以分离和鉴定与感兴趣的蛋白质相互作用的RNA。由于一些lncRNA是染色质的重要调节因子，因此该方法将被应用于研究与特定染色质蛋白相互作用的RNA。这些共价标记策略被设计为广泛适用，补充现有技术，并加速我们将调控RNA整合到重要的调控网络中。

DESCRIPTION (provided by applicant): A molecular-level understanding of regulatory networks has driven biomedical progress, but has mostly focused on protein networks. Non-coding RNAs (ncRNAs), including large ncRNAs (lncRNAs), also play important roles. For example, deletion of the Xist lncRNA causes cancers in mice. Yet the scope of lncRNA functions, their mechanisms of action, and their impact on therapeutically important pathways remain largely unknown. Approaches that identify high-stoichiometry or stable RNA-protein interactions have provided powerful insight into RNA, but miss important interactions, such as RNA helicase interactions with their substrates, that are transient or low stoichiometry. Similarly lncRNAs expressed at low abundance are important to regulation, yet the proteins they interact with are largely unknown. To address these needs, we are developing a platform to engineer covalent RNA modifications leading to new methodologies. First, the development of a covalent RNA tag will facilitate discovery of proteins that interact with low abundant lncRNAs. Second, this same platform is being used to develop a covalent-tracking strategy where a protein of interest is engineered to leave a covalent mark on transiently bound RNAs. This covalent modification will serve as a purification handle to isolate and identify RNAs that interacted with the protein of interest. As some lncRNAs are important regulators of chromatin, this approach will be applied to study RNAs that interact with specific chromatin proteins. These covalent-tagging strategies are designed to be broadly applicable, complement existing techniques, and accelerate our integration of regulatory RNAs into important regulatory networks.