Process-specific alternative splicing: a tool to monitor multiple alternative splicing events simultaneously in targeted plant genes

大多数植物基因含有插入序列（内含子）。当一个基因被打开（转录）时，DNA代码被复制到一个称为前体信使RNA（pre-mRNA）的RNA分子中。内含子序列通过剪接过程从前体mRNA中去除，剪接过程将基因的编码区（外显子）连接在一起。然后，剪接的mRNA被翻译成蛋白质。在许多情况下，在植物和动物中，前体mRNA可以以不同的方式剪接以产生不同的mRNA/这被称为选择性剪接（AS）。产生的替代mRNA可以编码具有不同功能的不同蛋白质，例如，在人类中，基因组中的35，000个基因可以产生超过150，000种蛋白质。因此，AS调节基因功能和表达，并增加高等真核生物中蛋白质的数量。这种灵活性使生物体中的细胞能够微调和微妙地调节细胞活性。AS不是一个随机过程，而是通过大量称为剪接因子的蛋白质与前mRNA中的序列信号相互作用而高度调节。因此，在特定的细胞类型中，剪接因子的谱将决定所有被表达的基因的选择性剪接转录物的模式。这将在不同的细胞类型中，在不同的发育阶段以及对刺激和例如应激条件的反应中有所不同。为了了解基因表达在选择性剪接水平上的调控，有必要能够测量多个基因在不同条件下选择性剪接的变化。在过去的五年里，估计植物基因的数量进行选择性剪接已经从7%上升到35%。尽管至少有三分之一的植物基因是选择性剪接的，但人们对植物中选择性剪接是如何调控的知之甚少。特别是，有必要更好地评估AS及其后果，以解决在同一生物过程中所涉及的基因中AS的协调调节，并能够检查细胞和组织特异性选择性剪接。目前的主要缺点之一是缺乏能够同时监测多个（10秒至100秒）AS事件的准确且可再现的系统。动物系统中的研究表明，选择性剪接是基因表达的一个重要方面，选择性剪接调控网络叠加在转录调控网络上。在植物系统中，常规地进行测量全局转录水平（转录组学），但是选择性剪接，特别是协调和调节的选择性剪接的概念在很大程度上被忽视。我们希望建立一个工具来监测多个植物基因在发育和胁迫反应中的选择性剪接的变化。该项目将在参与植物发育和应激生物学方面的小组与邓迪大学的RNA生物学/选择性剪接实验室之间建立合作。该项目的成果将证明，全面的特定工艺AS RT-PCR面板可用于准确分析不同条件下不同突变株系在发育过程中的可变剪接变化。通过关联特定基因或基因子集的选择性剪接的变化模式，将首次产生关于AS协调调节的信息。这些信息将是系统方法的一个组成部分，旨在了解调节生物过程的相互作用网络。我们将开发的工具将引起世界各地植物科学家的兴趣。虽然我们将通过研究拟南芥中的发育过程和胁迫诱导基因来建立该工具，但该系统非常灵活，可以应用于研究任何植物物种中存在合理EST数据的任何生物过程。

The majority of plant genes contain intervening sequences (introns). When a gene is turned on (transcribed), the DNA code is copied into a molecule of RNA called precursor messenger RNA (pre-mRNA). Intron sequences are removed from pre-mRNA by the process of splicing which joins the coding regions of genes (exons) together. The spliced mRNA is then translated into a protein. In many cases, in both plants and animals, pre-mRNAs can be spliced in different ways to generate different mRNAs / this is termed alternative splicing (AS). The alternative mRNAs produced can encode different proteins with different functions such that, for example, in humans, the 35,000 genes in the genome can give rise to more than 150,000 proteins. Thus, AS modulates gene function and expression and increases the number of proteins in higher eukaryotes. This flexibility allows the cells in an organism to fine-tune and subtly regulate cell activity. AS is not a random process but is highly regulated through the interaction of a large number of proteins called splicing factors with sequence signals in the pre-mRNA. Thus, in a particular cell type, the profile of splicing factors will determine the pattern of alternatively spliced transcripts of all of the genes being expressed. This will differ in different cell types, at different stages of development and in response to stimuli and, for example, stress conditions. To understand the regulation of gene expression at the level of alternative splicing, it is necessary to be able to measure changes in alternative splicing of multiple genes under different conditions. Over the last five years, estimates of the number of plant genes which undergo alternative splicing have risen from 7 to 35%. Despite at least a third of plant genes being alternatively spliced, little is known about how alternative splicing is regulated in plants. In particular, there is a need to better assess AS and its consequences, to address the co-ordinated regulation of AS in genes involved in the same biological process and to be able to examine cell- and tissue-specific alternative splicing. One of the major drawbacks currently is the lack of an accurate and reproducible system capable of monitoring multiple (10s to 100s) of AS events simultaneously. Research in animal systems has shown that alternative splicing is an essential aspect of gene expression with networks of alternative splicing regulation being superimposed on networks of transcriptional regulation. In plant systems, measuring global transcript levels is carried out routinely (transcriptomics) but alternative splicing and, in particular, the concept of co-ordinated and regulated alternative splicing has been largely ignored. We wish to establish a tool to monitor changes in alternative splicing of multiple plant genes in development and stress responses. The project will build collaborations between groups involved in aspects of developmental and stress biology of plants and the RNA biology/alternative splicing lab at the University of Dundee. The outcome of the project will be the demonstration that comprehensive process-specific AS RT-PCR panels can be used to accurately analyse changes in alternative splicing during development, under different conditions and in different mutant lines. By correlating patterns of changes in alternative splicing of specific genes or subsets of genes, information on the co-ordinated regulation of AS will be produced for the first time. Such information will be an integral part of systems approaches aimed at understanding interaction networks which regulate biological processes. The tool which we will develop will be of interest to plant scientists around the world. Although we will establish the tool by studying developmental processes and stress-induced genes in Arabidopsis, the system is very flexible and can be applied to examine any biological process in any plant species for which reasonable EST data exists.