Dynamic re-programming of the cold transcriptome in Arabidopsis

基因是遗传信息的储存库;蛋白质是执行活细胞功能的机器。基因表达通常是指基因产生蛋白质的过程。在真核生物中，基因表达是复杂的，并且当这样的蛋白质编码基因表达时，DNA序列首先通过转录复制成前体信使RNA（pre-mRNA）。前mRNA经历几个加工步骤以形成成熟的信使RNA（mRNA），其指导相应蛋白质的合成（翻译）。在这个项目中，我们专注于一个非常重要的RNA加工步骤，称为选择性剪接（AS）。AS从同一基因产生不同的mRNA转录物，从而可以调节转录物和蛋白质水平和功能。植物经历环境条件的不断变化，并进化出科普胁迫条件和在胁迫下生存的系统。在这个项目中，我们的重点是植物（拟南芥）对低温的反应和温度依赖的AS在植物对冷的反应中的作用。植物在任何特定时间的整体表达被称为转录组，它是所有正在表达的基因转录本的集合。它由1）转录（打开或关闭基因，向上或向下）和2）AS（从一个基因产生>1个转录本）决定。我们的高质量数据允许构建转录和AS网络。这将确定调节转录和AS反应的关键因素，以及当植物暴露于低温时转录组的重新编程。我们将检测这些基因对低温敏感性或耐受性的影响，从而确定提高作物耐寒性的候选基因。在这项研究中，我们将利用我们在选择性剪接和生物钟分析方面的专业知识，实现五个主要目标。(1)在我们已经获得的动态转录表达谱中，我们观察到许多基因在低温下的节律表达/AS的变化，包括失去或获得节律性的基因。这表明，生物钟可能参与这些基因的调节，通过分析在20 ℃和4 ℃下生长的植物的转录组，我们将确定那些基因，其改变的表达/AS是由时钟控制。(2)我们看到一些AS反应在冷却开始后非常迅速地发生，这表明预先存在的剪接因子（SF）的激活而不是新的剪接因子的形成，磷酸化是一种可能的机制。我们将调查即时/早期AS和SFs的磷酸化，以确定候选SFs可能参与这些快速反应冷却。(3)这项研究的一个主要部分将是表征新的冷反应基因，这些基因在对低温的反应中表现出显着的AS变化。将评估突变体、过表达和互补系的冷敏感性/耐受性和适应性。(4)我们现在能够生成高分辨率数据，使我们能够建立转录和剪接因子网络，并确定和验证调节冷诱导AS的关键SF（和转录因子）。这些数据还提供了整合转录和AS网络的机会，从而更好地了解转录组如何动态改变。(5)对于未来的网络建模，SF的RNA结合位点的知识是必不可少的。作为开发剪接密码的第一步，我们将对这里确定的关键SF的RNA结合位点进行测序。

Genes are the repositories of hereditary information; proteins are the machines that carry out the functions of living cells. Gene expression usually refers to the process by which a gene gives rise to a protein. In eukaryotes, gene expression is complex and when such protein-coding genes are expressed, the DNA sequence is first copied into a precursor messenger RNA (pre-mRNA) by transcription. The pre-mRNA undergoes several processing steps to form the mature messenger RNAs (mRNAs) which direct synthesis of the corresponding protein (translation). In this project we focus on an extremely important RNA processing step called alternative splicing (AS). AS generates different mRNA transcripts from the same gene and thereby can modulate transcript and protein levels and functions. Plants experience continual changes in environmental conditions and have evolved systems to cope with stress-causing conditions and to survive the stress. In this project we are focussed on the response of plants (Arabidopsis) to low temperature and the role of temperature-dependent AS in the plant response to cold. The overall expression of a plant at any particular time is called the transcriptome and it the collection of all of the gene transcripts being expressed. It is determined by 1) transcription (turning genes on or off, up or down) and 2) AS (making >1 transcript from a gene). The high quality of our data allows networks of transcription and AS to be constructed. This will identify key factors which regulate transcriptional and AS responses and the re-programming of the transcriptome when plants are exposed to low temperatures. We will characterise such genes for their effect on sensitivity or tolerance to low temperature thereby identifying candidate genes for improving cold tolerance in crop species.In this research we will address five main objectives by exploiting our expertise in alternative splicing and circadian clock analyses. (1) In the dynamic transcript expression profiles that we have already obtained, we observe changes in the rhythmic expression/AS of many genes at low temperature, including genes that lose or gain rhythmicity. This suggests that the circadian clock may be involved in the regulation of these genes and by analysing the transcriptomes of plants grown at 20C and 4C we will identify those genes whose altered expression/AS is controlled by the clock. (2) We see some AS responses that occur very rapidly after the onset of cooling, suggesting activation of pre-existing splicing factors (SFs) rather than formation of new ones, with phosphorylation as a likely mechanism. We will investigate immediate/early AS and phosphorylation of SFs to identify candidate SFs that may be involved in these rapid responses to cooling. (3) A major part of the research will be the characterisation of novel cold response genes which show significant AS changes in response to low temperature. Mutant, over-expression and complementation lines will be assessed for cold sensitivity/tolerance and acclimation.(4) We are now able to generate high resolution data enabling us to build transcription and splicing factor networks and identify and validate key SFs (and transcription factors) that regulate cold-induced AS. The data also gives the opportunity to integrate transcription and AS networks providing a much better understanding to how the transcriptome is dynamically altered.(5) For future network modelling, knowledge of RNA-binding sites of SFs is essential. We will characterise the RNA-binding sites of key SFs identified here as a first step to developing a splicing code.