Investigating the Role and Mechansims of miRNA Decay

描述（由申请人提供）：微小RNA（miRNAs）是高等真核生物基因调控的重要组成部分，引起靶mRNA的衰变或翻译抑制。miRNAs通常是动态表达的，并且一些有助于调节时空过程。因此，对这些主调节子的控制代表了基因调节的基本方面。尽管已经探索了miRNA上调的机制，但关于miRNA如何下调（动态表达的必要方面）知之甚少。先前已经提出，miRNA的下调主要由细胞生长和分裂期间的稀释介导。然而，这种机制不足以解释在许多反应中观察到的几种miRNAs水平的快速下降。为解决这一问题，本提案有两个具体目标。首先，我打算使用从同步化细胞中分离的小RNA的高通量测序来确定细胞周期期间miRNA的表达（Aim 1）。这项分析将使我能够确定在细胞周期中miRNA衰变和稀释的相对作用。此外，通过鉴定参与细胞周期的miRNA，该分析将扩展我们对它们在细胞周期和肿瘤发生中的作用的理解。第二，使用在Aim 1或之前在巨噬细胞刺激期间鉴定的不稳定的miRNA，我将研究miRNA衰变的分子机制（Aim 2）。我将通过敲除实验鉴定负责的核酸外切酶。然后通过分离天然复合物和随后的质谱法鉴定结合配偶体。为了鉴定miRNA不稳定性基序，将在整个miRNA上进行突变，然后将这些构建体的稳定性与内源性野生型miRNA的稳定性进行比较。该分析将结合生物信息学方法进行，以鉴定不稳定miRNA中的共同基序。减轻miRNA介导的抑制与诱导这种调节同样重要，因此理解miRNA下调的机制对于剖析多细胞生物中的基因调控至关重要。 公共卫生相关性：细胞具有相同的基因集合，但使它们不同的是哪些基因被打开，哪些基因被关闭。微小的RNA，称为microRNA，有助于做到这一点;当这些RNA分子不存在于适当的数量-太多或太少，-癌症有时会发展和/或生物体的发展可能会发生不正常。该提案的重点是了解microRNA的正确水平是如何控制和维持的。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are an important component of gene regulation in higher eukaryotes, eliciting either decay or translational repression of target mRNAs. miRNAs are often dynamically expressed, and some contribute to regulation of spatio-temporal processes. Control of these master regulators thus represents a fundamental aspect of gene regulation. Although mechanisms of miRNA up- regulation have been explored, little is known about how miRNAs are down-regulated, a necessary facet of dynamic expression. It has been previously proposed that down-regulation of miRNAs is primarily mediated by dilution during cell growth and division. However, such a mechanism is not sufficient to explain rapidly decreased levels of several miRNAs that have been observed during numerous responses. To address this issue, this proposal has two specific aims. First, I intend to determine miRNA expression during the cell cycle using high-throughput sequencing of small RNAs isolated from synchronized cells (Aim 1). This analysis will allow me to determine the relative roles of miRNA decay and dilution during the cell cycle. Moreover, by identifying miRNAs involved in the cell cycle, this analysis will extend our understanding of their role in the cell cycle and oncogenesis. Second, using unstable miRNAs identified either in Aim 1 or previously during stimulation of macrophages, I will investigate the molecular mechanisms of miRNA decay (Aim 2). I will identify the responsible exonuclease(s) by performing knock-down experiments. Binding partners will then be identified by isolating native complexes and subsequent mass spectrometry. To identify miRNA instability motifs, mutations will be made across the entire miRNA, and the stabilities of these constructs will then be compared with that of the endogenous, wild-type miRNA. This analysis will be performed in conjunction with bioinformatic approaches to identify common motifs in unstable miRNAs. Alleviation of miRNA-mediated repression is as important as induction of this regulation, and thus understanding the mechanisms by which miRNAs are down-regulated is critical to dissecting gene regulation in multicellular organisms. Public Health Relevance: Cells have the same collection of genes, but what makes them different is which genes are turned on and which are turned off. Tiny RNAs, called microRNAs, help do this; when these RNA molecules are not present in the proper numbers-either too many or too few,-cancer can sometimes develop and/or the development of an organism can occur improperly. This proposal focuses on understanding how the correct levels of microRNAs are controlled and maintained.