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

描述(申请人提供)：癌症、干细胞多能性和发育时机是LIN28在其中扮演主要角色的一些重要的生物学过程。当LIN28与OCT4、SOX2和NANOG联合使用时，可以诱导细胞重新编程到多潜能状态。Lin28的上调与细胞转化有关，许多人类肿瘤表现出高水平的Lin28(~15%)。作为涉及核因子-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基因变异与身高和青春期开始时间等人类发育特征的变异相关。LIN28是一种进化上保守的RNA结合蛋白，它抑制let-7家族的microRNAs。MicroRNAs是一种小的非编码RNA分子，调节特定的靶基因表达。为了产生功能成熟的microRNAs，前体RNA必须在初始转录后经过加工步骤。在这个转录后阶段，Lin28可以阻断(DROSHA和DICER)的两个切割步骤，并通过招募一个末端尿苷酰转移酶来促进降解，特别是对let-7 microRNAs。LIN28与let-7前体结合，但这些作用是如何实现的仍不清楚。此外，尽管Lin28的活性是let-7 microRNAs所特有的，但各种序列是如何被识别的也是未知的。为了从机制上了解Lin28的活性，还需要更多的分子细节来解释它的特异性和调控。我们将确定LiN28：LET-7络合物的高分辨结构以提供这样详细的信息。通过结合生化方法和结构方法，包括X射线结晶学和核磁共振光谱，我们将阐明Lin28是如何识别其靶标的。作为MYC的上游，LIN28是癌症治疗的一个有吸引力的目标。借助结构信息，我们将对LIN28的小分子抑制剂进行高通量搜索。LIN28的效应器功能也将通过研究它如何激活let-7前体上的尿酰转移酶来检测。我们的目标是了解Lin28如何具体结合其靶标，招募下游效应器，以及如何被阻断，这些都是更好地理解具有许多生物学意义的let-7调控的重要步骤。除了对控制let-7靶标的直接影响外，我们对Lin28--最早的microRNAs转录后调控因子之一--的了解可能适用于迅速增长的其他RNA结合因子清单，这些因子似乎调节其他miRNAs。最后，阐明Lin28是如何抑制microRNA加工的也将为microRNA加工中的中心事件的机制提供新的信息。 公共卫生相关性：let-7家族microRNAs的生物发生与许多关键的细胞事件有关，包括发育调节、控制多能性或肿瘤发生。LET-7microRNAs的成熟受LIN28调控，LIN28也受炎症信号通路调控。我们对let-7生物发生如何调控的结构性研究将使有用的研究工具的开发以及癌症治疗的潜在新途径成为可能。

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. PUBLIC HEALTH RELEVANCE: Biogenesis of let-7 family of microRNAs has been implicated in a number of key cellular events, including development regulation, controlling pluripotency, or tumorigenesis. Maturation of let-7 microRNAs is regulated by Lin28, which is also controlled by inflammation signaling pathways. Our structural investigation of how let-7 biogenesis is regulated will enable development of useful research tools as well as potential new avenues for cancer therapeutics.