Regulation of human microRNA biosynthesis and activity

人类 microRNA 生物合成和活性的调节

• 批准号: 6928633
• 负责人: BRYAN R. CULLEN
• 金额: $ 30.8万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6928633
• 关键词: RNA biosynthesis; RNA interference; cell differentiation; gene expression; genetic regulation; genetic translation; messenger RNA; molecular genetics; northern blottings; polymerase chain reaction

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are an extensive family of~21 nt long non-coding RNAs that are expressed in a wide range of eukaryotes, including humans. Current evidence suggests that miRNAs play a key role in differentiation and development by inhibiting the translation of mRNAs bearing partially complementary target sites. This effect has been reproduced in human cells in this laboratory using model mRNA targets and authentic but over-expressed human miRNAs. MiRNAs are closely related to small interfering RNAs (siRNAs), non-coding ~21 nt RNAs that can be introduced into cells as synthetic RNAs or derived from short hairpin RNAs (shRNAs) transcribed from expression plasmids. Although siRNAs usually function by inducing the degradation of mRNAs bearing fully complementary target sites, recent evidence suggests that siRNAs and miRNAs may function via similar mechanisms. Because of the likely importance of miRNAs in regulating gene expression, and the potential of siRNAs as artificial regulators of gene expression, it is important to understand how human miRNAs are made and how they function, if for no other reason than to permit the optimization of gene inactivation strategies that depend on siRNAs. In specific aim 1, we will attempt to understand how the initial miRNA transcript is precisely processed to yield a single, ~21 nt mature mRNA. Specifically, we will define the RNA sequences or structures that mediate the specific nuclear excision of the ~70 nt pre-miRNA intermediate from this initial transcript, a processing event very recently proposed to be mediated by human RNAse III/drosha. Moreover, we will define the features of this pre-miRNA that mediate the subsequent, equally specific cytoplasmic excision of the mature miRNA by dicer. In specific aim 2, we will extend our initial data showing that Exportin 5 (Exp5) is required for nuclear export of both pre-miRNAs and shRNAs, and that Exp5 binds the former specifically, by defining the characteristics of pre-miRNAs that mediate Exp5 binding. Specific aim 3 focuses on understanding how miRNAs cooperate to inhibit mRNA translation while specific aim 4 seeks to understood how endogenous miRNA stability and function is impacted by introduction of exogenous siRNAs. Finally, in specific aim 5, we seek to extend our exciting initial data demonstrating inhibition of miRNA function by the adenovirus VA1 non-coding RNA by trying to define the underlying mechanism.

描述（由申请人提供）：微小RNA（miRNA）是一个广泛的家族，长约21 nt，非编码RNA，在包括人类在内的广泛的真核生物中表达。目前的证据表明，miRNA通过抑制具有部分互补靶位点的mRNA的翻译在分化和发育中发挥关键作用。该实验室使用模型mRNA靶标和真实但过表达的人类miRNA在人类细胞中再现了这种效应。miRNA与小干扰RNA（siRNA）密切相关，小干扰RNA是非编码的~21 nt RNA，其可以作为合成RNA引入细胞或衍生自表达质粒转录的短发夹RNA（shRNA）。虽然siRNA通常通过诱导具有完全互补靶位点的mRNA的降解来发挥作用，但最近的证据表明siRNA和miRNA可能通过类似的机制发挥作用。由于miRNA在调节基因表达中的可能重要性，以及siRNA作为基因表达的人工调节剂的潜力，重要的是要了解人类miRNA是如何产生的以及它们是如何发挥作用的，如果没有其他原因，只为了允许优化依赖于siRNA的基因失活策略。在具体目标1中，我们将试图了解初始miRNA转录物如何精确加工以产生单个约21 nt的成熟mRNA。具体而言，我们将定义介导从该初始转录物中特异性核切除~70 nt前体miRNA中间体的RNA序列或结构，最近提出由人RNAse III/drosha介导的加工事件。此外，我们将定义这种pre-miRNA的特征，其介导随后的，同样特异性的成熟miRNA的细胞质切割。在具体目标2中，我们将通过定义介导Exp 5结合的pre-miRNA的特征来扩展我们的初始数据，该数据显示Exportin 5（Exp 5）是pre-miRNA和shRNAs的核输出所需的，并且Exp 5特异性地结合前者。具体目标3侧重于了解miRNA如何合作抑制mRNA翻译，而具体目标4则试图了解外源siRNA的引入如何影响内源性miRNA的稳定性和功能。最后，在具体的目标5中，我们试图通过定义潜在的机制来扩展我们令人兴奋的初始数据，这些数据证明了腺病毒VA 1非编码RNA对miRNA功能的抑制。