Temperature-Responsive Control of Splicing by RNA Methylation

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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.