transgenic expression of UPRT as a novel tool for tagging RNA in specific tissues of the mouse

摘要(Lay)RNA是利用DNA编码的信息合成的，它调节基因组的许多功能，并产生蛋白质。了解RNA的调节是理解细胞功能的首要任务。与DNA序列一样，RNA序列由四个碱基组成，但与DNA不同的是，RNA含有由嘧啶尿嘧啶组成的核苷尿苷。尿嘧啶没有被结合到老鼠或人类细胞的RNA中，因为这一途径所需的酶在哺乳动物基因组中没有编码。因此，控制表达的尿嘧啶磷酸核糖转移酶(UPRT)将尿嘧啶转化为尿苷一磷酸，从而允许尿嘧啶以细胞类型特异的或时间控制的方式标记RNA。我们已经产生了一种以可控的方式表达UPRT的转基因小鼠，我们想要证明它作为一种工具的广泛用途，用于纯化RNA或识别复杂细胞混合物中稀有细胞亚群中的RNA蛋白质相互作用。这是一项改进，因为它将打开获取某些细胞类型的RNA组成的途径，这些细胞类型不容易纯化。此外，已知的纯化行为会改变细胞内RNA的数量和质量。因此，通过减少操作次数，将获得更准确的RNA含量或RNA-蛋白质相互作用的读数。

Summary (Lay)RNA is synthesised using information encoded by DNA and it mediates many of the functions of the genome as well as producing proteins. Understanding the regulation of RNA is a priority for the understanding of cell function. Like DNA sequences, RNA sequences are made up for four bases, but, unlike DNA, RNA contains the nucleoside uridine which is composed of the pyrimidine uracil. Uracil is not incorporated into the RNA of mouse or human cells as the required enzymes for this pathway are not encoded in the mammalian genome. Consequently, controlled expression of the enzyme uracil phosphoribosyltransferase (UPRT) which converts uracil into uridine monophosphate allows the incorporation of uracil in cell-type specific or temporally controlled manner labelling of RNA. We have generated a transgenic mouse that expresses UPRT in a controllable way and we want to demonstrate the broad utility of this as a tool for purification of RNA or the identification of RNA protein interactions in rare cell subsets in complex mixtures of cells. This is an improvement because it will open access to the RNA makeup of some cell types that cannot be easily purified. Furthermore, the act of purification is known to change the quantity and quality of the RNA within the cell. Thus by reducing the numbers of manipulations a more accurate readout of RNA content or RNA-protein interactions will be obtained.