Investigating the Role and Mechansims of miRNA Decay

描述(申请人提供)：在高等真核生物中，microRNAs(MiRNAs)是基因调控的重要组成部分，可引起目标mRNAs的衰退或翻译抑制。MiRNAs通常是动态表达的，其中一些有助于调节时空过程。因此，对这些主调节器的控制代表了基因调控的一个基本方面。虽然miRNA上调的机制已经被探索，但对于miRNAs是如何下调的知之甚少，这是动态表达的一个必要方面。以前有人提出，miRNAs的下调主要是通过细胞生长和分裂过程中的稀释来调节的。然而，这样的机制不足以解释在许多反应中观察到的几个miRNAs水平的迅速下降。为了解决这个问题，这项提议有两个具体目标。首先，我打算使用从同步化细胞分离的小RNA的高通量测序来确定miRNA在细胞周期中的表达(目标1)。这一分析将使我能够确定miRNA衰退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miRNAs，这一分析将扩大我们对它们在细胞周期和肿瘤发生中的作用的理解。其次，利用目标1或之前在刺激巨噬细胞过程中发现的不稳定的miRNA，我将研究miRNA衰退的分子机制(目标2)。我将通过进行击倒实验来确定负责的核酸外切酶(S)。然后将通过分离天然络合物和随后的质谱分析来确定结合伙伴。为了识别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的稳定性进行比较。这一分析将与生物信息学方法一起进行，以确定不稳定miRNAs中的共同基序。MiRNA介导的抑制的缓解与这种调控的诱导同样重要，因此了解miRNAs下调的机制对于剖析多细胞生物体中的基因调控至关重要。 与公共卫生相关：细胞有相同的基因集合，但它们的不同之处在于哪些基因被打开，哪些被关闭。微小的RNA，称为microRNAs，有助于做到这一点；当这些RNA分子没有以适当的数量存在--无论是太多还是太少--癌症有时会发展和/或生物体的发育可能不正常。这项建议的重点是了解如何控制和维持正确的microRNAs水平。

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.