Temperature-Responsive Control of Splicing by RNA Methylation

最近气候变化影响的最明显的生物学例子之一是植物开花时间的改变。植物控制它们的开花时间，以确保它们在有利的条件下繁殖，它们利用环境温度作为这种控制的重要线索。许多英国物种现在比50年前开花更早，这对生态和农业产生了负面影响。然而，这方面的开花时间控制知之甚少，并提出了一个基本问题：植物如何测量温度？植物发育的控制依赖于植物染色体DNA编码的不同基因的作用。当基因被打开时，它们被复制到一种叫做RNA的相关分子中，RNA被翻译成基因编码的蛋白质。重要的是，DNA和RNA的拷贝在许多方面是不同的。一个关键的区别是DNA拷贝的延伸，称为内含子，必须精确地从RNA拷贝中切除，以使其正确地翻译成蛋白质。我们和其他人发现，从参与开花时间控制的基因中切除特定的内含子是温度敏感的。为了了解内含子的切除是如何受温度控制的，我们筛选了突变植物，以发现温度响应性控制开花不起作用的情况。我们发现了一个内含子去除不再对温度敏感的例子。这些植物有一个叫做FIONA的基因被破坏了，这个基因之前被一个来自韩国的群体发现会影响开花时间（FIONA在韩语里的意思是开花）。FIONA基因编码一种酶，这种酶通过在RNA序列的部分上添加甲基来对RNA进行化学修饰。通过对FIONA不起作用的植物的所有RNA进行测序，我们发现它们在切除特定内含子方面存在问题，包括已知以温度响应方式切除的内含子。DNA的一个著名特征是两条链通过碱基配对形成双螺旋结构。RNA与DNA的不同之处在于只有一条单链，但它倾向于尝试与自己配对，在这个过程中形成不同的形状和结构。FIONA结合到一个特定的RNA结构上，它添加的甲基影响碱基对的形成。重要的是，RNA结构对温度很敏感——它们在较低的温度下稳定，在较高的温度下融化。这种RNA温度计控制着细菌中的蛋白质翻译，最近在植物中也被证明具有同样的作用。我们的想法是，一种全新的RNA温度计创造了一种温度敏感的内含子RNA结构，由FIONA调节，以控制许多植物基因中内含子的温度敏感切除。为了验证这个想法，我们将仔细观察模式植物拟南芥中的RNA。首先，我们将确定FIONA与植物细胞中的哪些rna结合。然后，我们将使用一种称为纳米孔直接RNA测序的新测序技术来揭示不同温度下含有内含子的RNA的形状，绘制FIONA修饰的甲基RNA的图谱，并揭示当FIONA不在时，内含子切除在不同温度下的变化。通过这种方式，我们将把FIONA的行为与FIONA的行为联系起来。我们将仔细设计实验来测试FIONA如何控制从一个我们知道控制开花时间的基因中去除内含子，并将这一分析扩展到其他使用FIONA在不同温度下工作的基因。我们将把我们对rna的整体看法与这些详细的实验结合起来，以回答是什么使内含子的切除对温度敏感的基本问题。我们希望发现植物使用的新温度计。这项研究将让我们看到如何减轻气候变化对包括农作物在内的植物的影响。例如，我们可以重新设计RNA响应的温度，或者使用RNA温度计来控制其他重要的过程，如谷物发育。

One of the clearest biological examples of the impact of recent climate change has been the shift in the time at which plants flower. Plants control their time of flowering to ensure that they reproduce in favourable conditions and they use ambient temperature as an important cue for this control. Many UK species now flower earlier than they did fifty years ago and this has negative effects on ecology and agriculture. However, this aspect of flowering time control is poorly understood and raises the basic question: how do plants measure temperature?The control of plant development depends on the action of different genes encoded within the DNA of plant chromosomes. When genes are switched on, they are copied into a related molecule called RNA, which is translated into the protein coded by the gene. Importantly, the DNA and RNA copies differ in a number of respects. One crucial difference is that stretches of the DNA copy, called introns, must be precisely excised from the RNA copy to allow it to be correctly translated into protein. We, and others, found that excision of specific introns from genes involved in flowering time control is temperature-sensitive. To understand how the excision of introns is controlled by temperature, we screened mutant plants to find cases where temperature-responsive control of flowering did not work. We found one case where intron removal was not temperature-sensitive anymore. These plants had a disruption in a gene called FIONA, which had previously been found to affect flowering time by a group from South Korea (Fiona means flowering in Korean). The FIONA gene encodes an enzyme that chemically modifies RNA by adding a methyl group to parts of the RNA sequence. By sequencing all the RNA from plants where FIONA was not working, we found that they have a problem in excising specific introns, including introns known to be excised in a temperature-responsive manner.One of the famous features of DNA is that two strands form a double helix by base-pairing. RNA differs from DNA by having only a single strand, but it tends to try to base-pair with itself, making different shapes and structures in the process. FIONA binds to a specific RNA structure and the methyl group it adds influences whether base-pairs form. Importantly, RNA structures are sensitive to temperature - they are stabilised at lower temperature and melt at warmer temperatures. Such RNA thermometers control protein translation in bacteria and have recently been shown to do the same in plants. Our idea is that a completely novel type of RNA thermometer creates a temperature-sensitive intron RNA structure, modulated by FIONA, to control temperature-sensitive excision of introns in many plant genes.To test this idea, we will look carefully at RNA in the model plant, Arabidopsis. First, we will identify which RNAs in a plant cell, FIONA binds to. Then we will use a new sequencing technique called nanopore direct RNA sequencing to reveal the shapes of RNAs with introns at different temperatures, map the RNAs FIONA modifies with methyl groups and reveal how the intron excision changes at different temperatures when FIONA is not there. In this way, we will link where FIONA acts to what FIONA does. We will carefully design experiments to test how FIONA controls the excision of an intron from a gene that we know controls flowering time and expand this analysis to other genes that use FIONA to work at different temperatures. We will combine our global view of RNAs, with these detailed experiments to answer the fundamental question of what makes the excision of introns temperature sensitive. We hope to uncover new thermometers used by plants. This study will give us line of sight to how we might mitigate the impacts of climate change on plants, including crops. For example, we may be able to re-engineer the temperatures to which RNAs respond or use RNA thermometers to control other important processes like grain development.