Identification and characterisation of fip37-4 suppressor mutants in Arabidopsis thaliana

Bridgen, Alexander John / AJB (2022) Identification and characterisation of fip37-4 suppressor mutants in Arabidopsis thaliana. PhD thesis, University of Nottingham.

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Post-transcriptional and co-transcriptional RNA modifications play essential roles in controlling mRNA fate. The most abundant internal mRNA modification in eukaryotic cells is N6-methyladenosine (m6A) which has been shown to be critical for development in many eukaryotes and plays an important role influencing biological processes through its regulatory effects on RNA metabolism and translation. m6A is a dynamic modification with deposition of m6A on mRNA being catalysed by the methyltransferase “writer” complex, removed by demethylases “erasers” and recognised by m6A binding proteins known as “readers” which play specific regulatory roles. The Arabidopsis thaliana core m6A writer complex comprises of MTA (METTL3), MTB (METTL14), FIP37 (WTAP) VIRILIZER (VIRMA), HAKAI, HIZ1 and HIZ2 (likely homologue of ZC3H13). How m6A methylation machinery is regulated and knowledge of components mediating its various functions is still far from fully understood: especially in plants.

To further understand how m6A methylation is regulated and identify novel factors involved, a forward genetic suppressor screen was performed on hypomorphic FIP37 T-DNA insertion mutant fip37-4. A number of fip37-4 suppressor mutants were identified which exhibited wild-type (WT) like phenotypes. Further characterisation of the recessive suppressor mutant P2-04 demonstrated increased global m6A methylation to 57% WT-level, partial loss of fip37-4 developmental defects including hyperbranched trichomes, delayed flowering, reduced root growth, insensitivity to auxin and RT-qPCR analysis showed three-fold increase in functional FIP37 transcript. Bulked segregant analysis combined with comprehensive screening of backcrossed populations identified P2-04 causal mutation to likely be a missense mutation within the fourth KH domain of KH domain RNA binding protein AtKH26 (AtKH26R472Q). Confocal microscopy revealed AtKH26 to be nuclear localised and complementation of P2-04 with AtKH26-GFP transgene rescued fip37-4 phenotype suggesting AtKH26R472Q is the causal mutation. Transcriptomic analysis on P2-04 highlighted restored WT expression of genes differentially expressed (DE) in fip37-4 and several DE genes involved in auxin homeostasis and flowering possibly explaining P2-04 near WT phenotype. To further understand how AtKH26R472Q rescues fip37-4 T-DNA insertion mutant atkh26 was characterised identifying AtKH26 as a negative regulator of root development, flowering and m6A measurements showed m6A levels to be increased by 5%. Crossing atkh26 with hypomorphic VIRILIZER mutant vir-1 failed to completely suppress vir-1 however m6A levels and root length were increased and displayed earlier flower therefore suggesting complete suppression by mutating AtKH26 is specific to fip37-4. Protein-RNA affinity assay with m6A modified RNA oligos demonstrated both AtKH26 and AtKH26R472Q to preferentially bind to non-modified RNA concluding AtKH26 as an anti-m6A reader and must negatively regulate m6A containing transcripts through some alternative mechanism.

Overall, this study provides insight on AtKH26 as a novel KH domain protein involved in regulating plant development and FIP37 directed m6A methylation: likely playing a role in nuclear metabolism of FIP37 mRNA and transcripts involved in root and floral development.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Fray, Rupert / RGF
Seymour, Graham / GBS
Keywords: fip37, m6a, RNA, Arabidopsis, KH domain, flowering, root, auxin, virilizer, AtKH26, EMS, trichome, methylation, epitranscriptome, epitranscriptomics
Subjects: Q Science > QK Botany > QK457 Spermatophyta. Phanerogams
Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 71522
Depositing User: Bridgen, Alexander
Date Deposited: 14 Dec 2022 04:40
Last Modified: 14 Dec 2022 04:40

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