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

Lin28调控let-7生物发生的结构和机制研究

• 批准号: 8218831
• 负责人: Piotr Sliz
• 金额: $ 35.07万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8218831
• 关键词: Affect; Affinity; Binding; Binding Sites; Biochemical; Biogenesis; Biological; Biological Assay; Biological Process; Cells; Chemicals; Complex; Couples; Crystallography; Data; Development; Docking; Drug Design; Enzymes; Evaluation; Event; Exhibits; Family; Feedback; Fluorescein; Fluorescence Polarization; Functional RNA; Gene Expression; Genes; Genetic Transcription; Goals; Height; Human; In Vitro; Inflammation; Investigation; Label; Learning; Libraries; Malignant Neoplasms; Methods; MicroRNAs; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; NF-kappa B; NMR Spectroscopy; Nucleotides; Play; Positioning Attribute; Precursor RNA; Process; Proteins; Puberty; RNA; RNA Binding; RNA-Binding Proteins; Recombinants; Recruitment Activity; Regulation; Relative (related person); Research; Resolution; Role; Signal Pathway; Site; Solutions; Specificity; Staging; Structure; Testing; Therapeutic; Time; Transferase; UDPglucose-Hexose-1-Phosphate Uridylyltransferase; Up-Regulation; Validation; Variant; Vertebral column; X-Ray Crystallography; base; cancer stem cell; cell transformation; design; high throughput screening; human DICER1 protein; inhibitor/antagonist; member; paralogous gene; pluripotency; small molecule; tool; trait; tumor; tumorigenesis

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.

描述（由申请人提供）：癌症、干细胞多能性和发育时机是Lin 28发挥主要作用的一些重要生物学过程。Lin 28在与OCT 4、SOX 2和NANOG组合使用时可以诱导细胞重编程为多能状态。Lin 28的上调与细胞转化相关，许多人类肿瘤显示出高水平的Lin 28（~15%）。作为涉及NF-κ B的反馈回路的一部分，Lin 28将炎症和细胞转化偶联。此外，Lin 28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。Lin 28是一种进化上保守的RNA结合蛋白，可抑制let-7家族的microRNA。MicroRNA是一种非编码RNA小分子，可调控特定靶基因的表达。为了产生功能成熟的微小RNA，前体RNA必须在初始转录后经历加工步骤。在该转录后阶段，Lin 28可以阻断两个切割步骤（通过Drosha和Dicer），并通过募集末端尿苷酰转移酶促进降解，特别是对于let-7 microRNA。lin 28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Lin 28的活性对let-7 microRNA具有特异性，但各种序列如何被识别也是未知的。为了获得对Lin 28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节。我们将确定Lin 28：let-7复合物的高分辨率结构以提供这些详细信息。通过结合生物化学方法和结构方法，包括X射线晶体学和NMR光谱学，我们将阐明Lin 28如何识别其靶标。作用于MYC的上游，Lin 28成为癌症治疗的有吸引力的靶标。在结构信息的帮助下，我们将进行高通量搜索Lin 28的小分子抑制剂。还将通过研究Lin 28如何激活let-7前体上的尿苷酰转移酶来检查Lin 28的效应子功能。我们的目标是了解Lin 28如何特异性地结合其靶标，招募下游效应子，并且可以被阻断，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Lin 28的了解-microRNA的第一个转录后调节因子之一-可能适用于快速增长的其他RNA结合因子列表，这些因子似乎可以调节其他miRNA。最后，阐明Lin 28如何抑制microRNA加工也将为microRNA加工中的中心事件机制提供新的信息。 公共卫生相关性：let-7家族microRNA的生物发生涉及许多关键细胞事件，包括发育调节、控制多能性或肿瘤发生。let-7 microRNA的成熟受Lin 28调节，Lin 28也受炎症信号通路控制。我们对let-7生物合成如何调控的结构研究将有助于开发有用的研究工具以及癌症治疗的潜在新途径。