Presentation Information
[P03-322]Design of a highly active sequence-specific RNA modification tool with low affinity RNA binding protein
○Azumi Uchida1, Shiroh Futaki2, Miki Imanishi1 (1. Institute for Chemical Research, Kyoto Univ. (Japan), 2. Grad. Sch. Pharm. Sci., Kyoto Univ. (Japan))
Keywords:
RNA modification,Enzyme,kinetics,epigenome
[Purpose]
N6-methylation of adenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes and is involved in various important biological processes such as development and circadian rhythm. Therefore, elucidating the precise functions of m6A is of critical importance. To control the levels of methylation of individual adenosine, our lab developed FTO-PUF, a fusion protein of RNA binding protein PUF and an m6A demethylase FTO [1]. Additionally, to suppress off-target effects at high enzyme concentration, we developed modulated FTO-PUF (modFTO-PUF) by introducing mutations into FTO [2]. However, the relationship between RNA recognition by PUF and the demethylation activity of the fusion protein, modFTO-PUF, remains unknown. In this study, we aimed to improve demethylation activity of modFTO-PUF by optimizing the binding affinity between PUF and its target RNA.
[Method]
To decrease the binding affinity between PUF and RNA, we designed a mutant PUF (mutPUF) by introducing mutations into amino acids residues involved in RNA recognition. The resulting mutPUF was fused to modFTO to generate modFTO-mutPUF. The binding parameters of modFTO-wtPUF and modFTO-mutPUF to the target RNA were evaluated by measuring their association rate constant (ka) and dissociation rate constant (kd) using Bio-Layer Interferometry (BLI). Furthermore, their enzymatic activities defined as the amount of demethylated target adenosine, and their turnover numbers (kcat) were assessed using the m6A-sensitive RNA endonuclease MazF.
[Results]
BLI analysis revealed that the dissociation rate constant (kd) of modFTO-mutPUF was significantly higher, whereas the association rate constant (ka) showed only small changes, demonstrating that the binding affinity of modFTO-mutPUF was lower than that of modFTO-wtPUF. Despite the lower affinity, both the turnover number and the demethylation activity of modFTO-mutPUF were higher than those of modFTO-wtPUF.
[Consideration]
These results suggest that the mutations introduced into PUF induced faster dissociation of modFTO-mutPUF from target RNA, leading to increase in the turnover number, and enhancement of sequence-specific demethylation activity.
[Conclusion]
This study demonstrated that the enzymatic activity of RNA-targeting demethylation tools can be improved by fine-tuning the binding affinity between RNA binding protein and target RNA. This tool would contribute to regulate the rapid changes in the m6A levels during cell cycles and circadian rhythms, and to clarify the importance of m6A in biological systems.
[References]
[1] K. Shinoda et al., Chem. Commun., 2020, 56, 1365-1368
[2] K. Otonari et al., Chem. Commun., 2025, 61, 69-72
N6-methylation of adenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes and is involved in various important biological processes such as development and circadian rhythm. Therefore, elucidating the precise functions of m6A is of critical importance. To control the levels of methylation of individual adenosine, our lab developed FTO-PUF, a fusion protein of RNA binding protein PUF and an m6A demethylase FTO [1]. Additionally, to suppress off-target effects at high enzyme concentration, we developed modulated FTO-PUF (modFTO-PUF) by introducing mutations into FTO [2]. However, the relationship between RNA recognition by PUF and the demethylation activity of the fusion protein, modFTO-PUF, remains unknown. In this study, we aimed to improve demethylation activity of modFTO-PUF by optimizing the binding affinity between PUF and its target RNA.
[Method]
To decrease the binding affinity between PUF and RNA, we designed a mutant PUF (mutPUF) by introducing mutations into amino acids residues involved in RNA recognition. The resulting mutPUF was fused to modFTO to generate modFTO-mutPUF. The binding parameters of modFTO-wtPUF and modFTO-mutPUF to the target RNA were evaluated by measuring their association rate constant (ka) and dissociation rate constant (kd) using Bio-Layer Interferometry (BLI). Furthermore, their enzymatic activities defined as the amount of demethylated target adenosine, and their turnover numbers (kcat) were assessed using the m6A-sensitive RNA endonuclease MazF.
[Results]
BLI analysis revealed that the dissociation rate constant (kd) of modFTO-mutPUF was significantly higher, whereas the association rate constant (ka) showed only small changes, demonstrating that the binding affinity of modFTO-mutPUF was lower than that of modFTO-wtPUF. Despite the lower affinity, both the turnover number and the demethylation activity of modFTO-mutPUF were higher than those of modFTO-wtPUF.
[Consideration]
These results suggest that the mutations introduced into PUF induced faster dissociation of modFTO-mutPUF from target RNA, leading to increase in the turnover number, and enhancement of sequence-specific demethylation activity.
[Conclusion]
This study demonstrated that the enzymatic activity of RNA-targeting demethylation tools can be improved by fine-tuning the binding affinity between RNA binding protein and target RNA. This tool would contribute to regulate the rapid changes in the m6A levels during cell cycles and circadian rhythms, and to clarify the importance of m6A in biological systems.
[References]
[1] K. Shinoda et al., Chem. Commun., 2020, 56, 1365-1368
[2] K. Otonari et al., Chem. Commun., 2025, 61, 69-72
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