Award details

Temperature Responsive Control of Splicing by RNA Methylation

Reference	BB/W007673/1
Principal Investigator / Supervisor	Professor Gordon Simpson
Co-Investigators / Co-Supervisors
Institution	University of Dundee
Department	School of Life Sciences
Funding type	Research
Value (£)	363,689
Status	Current
Type	Research Grant
Start date	19/09/2022
End date	18/09/2025
Duration	36 months

Abstract

There is no mechanism to explain how the precise measurement of temperature could be linked to splicing decisions. This hinders our ability to understand how plants respond to temperature and also prevents us from using this versatile system to engineer artificial temperature-responsive splicing events, for synthetic biology and agriculture. Using a mutant screen and nanopore direct RNA sequencing, we discovered that the m6A RNA methyltransferase, FIO1, confers the property of temperature-responsiveness onto multiple splice events, some of which are already known to respond to temperature changes. The human orthologue of FIO1 is METTL16, which binds RNA stem-loop structures to control splicing and methylates noncoding RNAs, including U6snRNA. m6A base modifications alter RNA base-pair formation in context-dependent ways. Together, these findings suggest that plants utilise RNA methylation and thermosensitive RNA-RNA interactions, via a novel type of RNA thermometer, to control splicing. We seek to understand how FIO1 recognises its target RNAs, how it modifies them and how this is linked to RNA processing. We will use state-of the-art methods to map the exact binding sites for FIO1 in nuclear RNA across a range of physiologically relevant temperatures and to probe the secondary structure of nuclear RNAs in the same conditions. We will use nanopore direct RNA sequencing to discover the sites of FIO1-dependent m6A modification and the FIO1-dependent splicing events, at the same temperatures. Through in vitro RNA binding experiments, with purified recombinant FIO1, and in vivo assays, by mutating temperature-responsive introns, we will determine the mechanism by which m6A, temperature and RNA structure interact. By making the corresponding mutations on MAF2 transgenes we will link the mechanism to the biology of flowering time control. We will test our models by engineering artificial biological "thermostats" to express transgenes in specific temperature ranges.

Summary

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 doublehelix 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.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Plant Science, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/W007967/1 Temperature-Responsive Control of Splicing by RNA Methylation

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