Structural and Mechanistic Studies of Regulation of let-7 biogenesis by Lin28'

描述（由申请人提供）：癌症、干细胞多能性和发育时序是Lin28在一些重要的生物学过程中起主要作用。当Lin28与OCT4、SOX2和NANOG联合使用时，可以诱导细胞重编程到多能状态。Lin28的上调与细胞转化有关，许多人类肿瘤显示高水平的Lin28（~15%）。作为涉及NF- kB的反馈回路的一部分，Lin28将炎症和细胞转化结合在一起。此外，Lin28变异与人类发育特征的变异相关，如身高和青春期开始的时间。Lin28是一种进化上保守的rna结合蛋白，可抑制let-7家族的microRNAs。MicroRNAs是调节特定靶基因表达的小的非编码RNA分子。为了产生功能成熟的microrna，前体RNA在初始转录后必须经过加工步骤。在这个转录后阶段，Lin28可以阻断两个裂解步骤（由Drosha和Dicer完成），并通过募集末端脲基转移酶促进降解，特别是对于let-7 microRNAs。Lin28与let-7前体结合，但这些作用是如何实现的尚不清楚。此外，尽管Lin28活性对let-7 microrna是特异性的，但各种序列如何被识别也是未知的。为了获得对Lin28活性的机制理解，需要更多的分子细节来解释其特异性和调控。我们将确定Lin28:let-7配合物的高分辨率结构来提供这些详细的信息。通过结合生化方法和结构方法，包括x射线晶体学和核磁共振波谱，我们将阐明Lin28如何识别其目标。Lin28作用于MYC上游，是癌症治疗的一个有吸引力的靶点。在结构信息的帮助下，我们将对Lin28的小分子抑制剂进行高通量搜索。Lin28的效应功能也将通过研究它如何激活let-7前体上的尿苷基转移酶来研究。我们的目标是了解Lin28如何特异性结合其靶标，招募下游效应物，以及如何被阻断，这些都是更好地理解let-7调控的重要步骤，具有许多生物学意义。除了它对控制let-7靶点的直接影响外，我们对lin28的了解——最早的microrna转录后调节因子之一——可能应用于快速增长的其他rna结合因子列表，这些因子似乎调节其他microrna。最后，阐明Lin28如何抑制microRNA加工也将为microRNA加工中心事件的机制提供新的信息。

DESCRIPTION (provided by applicant): Cancer, stem cell pluripotency, and developmental timing are some of the important biological processes that Lin28 plays a major role in. Lin28 can induce cell reprogramming to pluripotent state when used in combination with OCT4, SOX2, and NANOG. Up-regulation of Lin28 correlates with cell transformation, and many human tumors exhibit high levels of Lin28 (~15%). As part of a feedback loop that involves NF- kB, Lin28 couples inflammation and cell transformation. Moreover, Lin28 variations correlate with variance in human developmental traits such as height and timing of puberty onset. Lin28 is an evolutionarily conserved RNA-binding protein that inhibits the let-7 family of microRNAs. MicroRNAs are small non- coding RNA molecules that regulate specific target-gene expression. In order to generate functional mature microRNAs, the precursor RNA has to undergo processing steps after initial transcription. During this post- transcriptional stage, Lin28 can block both of the cleavage steps (by Drosha and Dicer), and promote degradation by recruiting a terminal uridylyl transferase, specifically for the let-7 microRNAs. Lin28 binds to let-7 precursors, but how these effects are achieved is still unclear. Moreover, although Lin28 activity is specific for let-7 microRNAs, how the various sequences are recognized is also unknown. In order to gain a mechanistic understanding of Lin28 activity, more molecular details are required to explain its specificity and regulation. We will determine high resolution structures of Lin28:let-7 complexes to provide such detailed information. By combining biochemical methods and structural approaches including X-ray crystallography and NMR spectroscopy, we will clarify how Lin28 recognizes its target. Acting upstream of MYC, Lin28 poses as an attractive target for cancer therapeutics. With the help of the structural information, we will perform a high-throughput search for a small molecule inhibitor of Lin28. The effector function of Lin28 will also be examined by investigating how it activates uridylyl transferase on let-7 precursors. Our goal of understanding how Lin28 specifically binds its target, recruits a downstream effector, and can be blocked, are all important steps to better understand let-7 regulation with many biological implications. In addition to its direct impact on controlling let-7 targets, what we learn about Lin28-one of the first post- transcriptional regulators of microRNAs-may be applied to a rapidly growing list of other RNA-binding factors that appear to regulate other miRNAs. Finally, elucidating how Lin28 inhibits microRNA processing will also provide new information on the mechanism of central events in microRNA processing.