Investigating the Role and Mechansims of miRNA Decay

描述（由申请人提供）：MicroRNAs （miRNAs）是高等真核生物基因调控的重要组成部分，可引起靶mrna的衰变或翻译抑制。mirna通常是动态表达的，其中一些有助于调节时空过程。因此，控制这些主调节器代表了基因调控的一个基本方面。虽然已经探索了miRNA上调的机制，但对miRNA如何下调知之甚少，而下调是动态表达的必要方面。先前已经提出mirna的下调主要是通过细胞生长和分裂过程中的稀释介导的。然而，这种机制不足以解释在许多反应中观察到的几种mirna水平的迅速下降。为了解决这个问题，这项建议有两个具体目标。首先，我打算利用从同步细胞中分离的小rna的高通量测序来确定细胞周期中miRNA的表达（目的1）。这一分析将使我能够确定miRNA衰减和稀释在细胞周期中的相对作用。此外，通过识别参与细胞周期的mirna，该分析将扩展我们对其在细胞周期和肿瘤发生中的作用的理解。其次，使用在Aim 1中或之前在巨噬细胞刺激过程中发现的不稳定miRNA，我将研究miRNA衰变的分子机制（Aim 2）。我将通过进行敲除实验来确定负责的外切酶。然后通过分离天然配合物和随后的质谱法鉴定结合伙伴。为了确定miRNA不稳定基序，将在整个miRNA中进行突变，然后将这些结构的稳定性与内源性野生型miRNA进行比较。该分析将与生物信息学方法一起进行，以确定不稳定mirna中的共同基序。减轻mirna介导的抑制与诱导这种调控同样重要，因此了解mirna下调的机制对于解剖多细胞生物中的基因调控至关重要。

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.