Role of microRNAs in the control of mRNA translation

本项目研究一类新发现的核糖核酸（RNA）分子的功能，称为microRNAs （miRNAs）。它们似乎在所有多细胞生物中都有非常重要的功能，因为它们中的许多在数亿年里几乎没有发生变化。目前，我们知道mirna可能通过调节信使rna （mrna）翻译成蛋白质分子的效率来起作用。然而，我们不知道它们是如何做到这一点的，也不知道哪些mrna是由mirna调节的。到目前为止，我们的研究表明，mirna可能会相互联合作用于mRNA，因此对特定mRNA的影响取决于存在哪些mirna，以及它们的丰度。我们建议构建一个可以在体外研究mirna功能的系统，使用已知具有mRNA翻译和相关功能的粗细胞提取物。这将首次使在严格控制的条件下测试mirna成为可能，并询问它们影响翻译的哪些步骤。我们将能够研究miRNA和mRNA序列的配对如何决定miRNA作用于特定mRNA的能力，并确认给定的miRNA实际上调节特定mRNA。为了实现这一目标，我们将使用三种方法分离作为miRNA调控靶点的mrna。这些方法都依赖于细胞提取物中miRNA和mrna的物理相互作用。在一种方法中，我们将在miRNA上附着一个标签，并使用该标签拔出相关的mrna。在另一种方法中，我们将使用一种被认为与所有mirna相关的蛋白质的抗体。抗体应该能提取出任何与mirna -蛋白复合物相关的mrna。最后，我们将通过改变翻译效率来识别对miRNA的存在做出反应的mrna。这些实验将提供关于一个极其重要的生物系统的基本知识。

This project studies the function of a recently discovered class of ribonucleic acid (RNA) molecules termed microRNAs (miRNAs). They appear to have very important functions in all multicellular organisms, since many of them have undergone little change over hundreds of millions of years. At present we know that miRNAs probably work by regulating the efficiency with which messenger RNAs (mRNAs) are translated into protein molecules. However we do not know how they accomplish this, or which mRNAs are regulated by miRNAs. Our work to date has suggested to us that miRNAs may act in combination with one another on mRNAs, so that the effect on a given mRNA depends on just which miRNAs are present, and how abundant they are. We propose to construct a system in which the function of miRNAs can be studied in vitro, using crude cell extracts that are known to perform mRNA translation and related functions. This will, for the first time, make it possible to test miRNAs in carefully controlled conditions, and ask which steps in translation they affect. We will be able to investigate just how the pairing of miRNA and mRNA sequences determines the ability of a miRNA to act on a specific mRNA, and to confirm that a given miRNA actually does regulate a specific mRNA. To facilitate this goal, we will use three methods to isolate mRNAs that are targets of miRNA regulation. Each of these methods relies on the physical interaction of miRNA and mRNAs in cell extracts. In one method, we will attach a tag to a miRNA, and use the tag to pull out associated mRNAs. In another method, we will use an antibody to a protein that is thought to associate with all miRNAs. The antibody should pull out any mRNAs associated with the miRNA-protein complex. Finally, we will identify mRNAs that have responded to the presence of a miRNA by changing the efficiency with which they are translated. These experiments will provide essential knowledge about an extremely important biological system.