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变化。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，它们将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年前更早开花，这对生态和农业都有负面影响。然而，人们对开花时间控制的这一方面知之甚少，并提出了一个基本问题：植物如何测量温度？植物发育的控制取决于植物染色体DNA中编码的不同基因的作用。当基因被激活时，它们被复制到一种名为RNA的相关分子中，该分子被翻译成由基因编码的蛋白质。重要的是，DNA和RNA拷贝在许多方面不同。一个关键的区别是，DNA复制的延伸部分，称为内含子，必须从RNA复制中精确地切除，才能使其正确地翻译成蛋白质。我们和其他人发现，从涉及开花时间控制的基因中剔除特定内含子是温度敏感的。为了了解温度是如何控制内含子的删除的，我们对突变植物进行了筛选，以找出对温度敏感的开花控制不起作用的情况。我们发现了一个内含子去除不再对温度敏感的案例。这些植物的一种名为Fiona的基因发生了突变，此前韩国的一个研究小组发现该基因会影响开花时间(Fiona在韩语中的意思是开花)。Fiona基因编码一种酶，通过在部分RNA序列中添加甲基来对RNA进行化学修饰。通过对菲奥娜没有工作的植物的所有RNA进行测序，我们发现它们在切除特定内含子时存在问题，包括已知以温度响应的方式切除的内含子。DNA的著名特征之一是两条链通过碱基配对形成双螺旋。RNA与DNA的不同之处在于它只有一条单链，但它倾向于尝试与自己碱基配对，在这个过程中形成不同的形状和结构。菲奥纳与特定的RNA结构结合，它添加的甲基会影响碱基对的形成。重要的是，RNA结构对温度很敏感--它们在较低的温度下稳定下来，在较温暖的温度下融化。这种RNA温度计控制细菌中的蛋白质翻译，最近已被证明在植物中也做了同样的事情。我们的想法是，一种全新类型的RNA温度计创建了一种受Fiona调控的温度敏感的内含子RNA结构，以控制许多植物基因中对温度敏感的内含子的切割。为了测试这一想法，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，我们将使用一种名为纳米孔直接RNA测序的新测序技术来揭示不同温度下带有内含子的RNA的形状，用甲基团映射Fiona修饰的RNA，并揭示当Fiona不在那里时，内含子切除在不同温度下的变化。通过这种方式，我们将把菲奥娜的行为与菲奥娜的所作所为联系起来。我们将仔细设计实验，以测试菲奥纳如何控制从我们已知的控制开花时间的基因中切除内含子，并将这一分析扩展到使用菲奥纳在不同温度下工作的其他基因。我们将结合我们对RNA的全球观点和这些详细的实验来回答是什么使内含子的切除对温度敏感这一根本问题。我们希望发现植物使用的新温度计。这项研究将让我们了解如何减轻气候变化对包括农作物在内的植物的影响。例如，我们可能能够重新设计RNA对其做出反应的温度，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.