Decoding the molecular principles underlying tumor-suppressive or oncogenic properties of large non-coding RNAs

解码大型非编码rna抑制肿瘤或致癌特性的分子原理。当前基因组生物学中最有趣的问题之一是大型非编码rna （lncRNAs）的功能作用。lncrna表现出显著的组织特异性表达、清晰的保守模式和基因样染色质特征。重要的是，单个lncrna正在成为肿瘤发生事件的强大调节因子，因此可以归类为肿瘤抑制因子或致癌基因。与其调控的重要性相反，对其致癌或肿瘤抑制特性的机制理解尚缺乏。揭示lncRNA功能的分子原理的一个关键先决条件是对其蛋白质结合伙伴的详细了解。在这里，我利用RNA反义纯化和定量质谱法来鉴定两种肿瘤抑制和两种致癌lncrna的直接蛋白质结合伙伴。在确定特定的蛋白质结合物后，我将使用交联和免疫沉淀来绘制它们的RNA结合位点。利用这些信息，我将系统地将捕获的蛋白质相互作用分为功能上必要和非必要的相互作用。我将突变lncrna内不同的蛋白质结合域，并监测已知的lncrna驱动的各自细胞致瘤效力的变化。因此，我将能够将lncRNA依赖的致瘤表型与每种lncRNA特异性募集的一组不同的蛋白质调节因子联系起来。本研究将阐明介导致癌或抑瘤lncRNA特性的分子成分，并进一步揭示这些调控因子的重要设计原理。利用这些见解，我将基于功能重要的蛋白质相互作用域构建合成的lncRNA替代结构，并测试这些结构是否保留所需的调节活性（例如肿瘤抑制活性）。我设想这种方法将为新的基于rna的治疗策略打开大门，并改变我们对lncrna介导的癌症生物学中基因调控的理解。

Decoding the molecular principles underlying tumor-suppressive or oncogenic properties of large non-coding RNAs. One of the most intriguing questions in current genome biology concerns the functional role of large non-coding RNAs (lncRNAs). LncRNAs show remarkable tissue specific expression, clear patterns of conservation and gene-like chromatin signatures. Importantly, individual lncRNAs are emerging as powerful regulators of tumorigenic events and can thus be classified as tumor suppressors or oncogenes. In contrast to their regulatory importance, mechanistic understanding of their oncogenic or tumor-suppressive properties is lacking. A key prerequisite to reveal the molecular principles underlying lncRNA function is the detailed knowledge of their protein-binding partners. Here, I utilize RNA antisense purification followed by quantitative mass spectrometry to identify the direct protein-binding partners of two tumor-suppressive and two oncogenic lncRNAs. Following identification of specific protein binders, I will use crosslinking and immunoprecipitation to map their RNA binding sites. Harnessing this information, I will systematically classify captured protein interactions into functionally essential and non-essential interactions. I will mutate distinct protein binding domains within lncRNAs and monitor known lncRNA-driven changes in the tumorigenic potency of respective cells. Thus, I will be able to connect lncRNA-dependent tumorigenic phenotypes to the specific recruitment of a distinct set of protein regulators to each lncRNA. This research will illuminate the molecular components that mediate oncogenic or tumor-suppressive lncRNA properties and further reveal important design principles of these regulators. Exploiting these insights, I will build synthetic lncRNA replacement constructs based on functionally important protein interaction domains and test if these constructs retain desired regulatory activities (e.g. tumor-suppressive activity). I envision that this approach will open the door for novel RNA-based therapeutic strategies and transform our understanding of lncRNA-mediated gene regulation in cancer biology.