Identification of factors required for m⁶A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

Abstract

Summary N6‐adenosine methylation (m6A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood.Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6A writer proteins in Arabidopsis thaliana.The components required for m6A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6A methylation plays a role in developmental decisions during pattern formation.The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

Topologies of N6‐adenosine methylation (m6A) in land plant mitochondria and their putative effects on organellar gene expression

“…Global RNA‐seq analyses of the model plant A. thaliana indicated the presence of m 6 A modifications within many nuclear‐encoded mRNA transcripts (Zhong et al , ; Bodi et al , ; Shen et al , ; Růžička et al , ; Wei et al , ; Parker et al , ). These analyses further suggested that the organellar (i.e.…”

“…The nuclear genome of A. thaliana contains several genes that are closely related to m 6 A methyltransferases (see for example Růžička et al , and Table S6). These include an MTA‐related protein (At4g10760) and an orthologue of the mammalian WTAP, FIP37 (encoded by the AT3G54170 gene locus), both of which were shown to play key roles in determining the m 6 A mRNA modification patterns in Arabidopsis (Zhong et al , ; Bodi et al , ; Luo et al , ; Shen et al , ; Růžička et al , ). Sequence similarity searches using PSI‐BLAST were used to identify putative components of the organellar m 6 A methyltransferase complex from Arabidopsis and rice genomes.…”

“…This was also supported by Nanopore direct RNA sequencing of Arabidopsis plants (Parker et al , ). The significance of m 6 A is apparent in mutant plants with reduced levels of m 6 A that show embryogenesis defects and altered growth and developmental phenotypes (Zhong et al , ; Bodi et al , ; Wan et al , ; Shen et al , ; Růžička et al , ; Arribas‐Hernández et al , ; Chen et al , ; Li et al , ; Wei et al , ). Global m 6 A‐RIP‐seq analyses also indicated the presence of m 6 A within organellar (i.e.…”

N6‐methyladenosine regulatory machinery in plants: composition, function and evolution

“…A number of proteins involved in the addition, removal and identification of m6A have been reported, which are categorized into three groups called writers, erasers and readers, respectively, and include proteins such as MTA, MTB, ALKBH9B and ECT2 (Figure ) (Arribas‐Hernández et al ., ; Fu et al ., ; Martínez‐Pérez et al ., ; Zhong et al ., ). The m6A writer complex recognizes the consensus motif RRACH (Růžička et al ., ). However, not all RRACH motifs are associated with m6A modification, as the m6A level is much lower than the abundance of RRACH motifs, indicating that the molecular mechanism regulating m6A modification is not fully understood (Liu and Pan, ).…”

“…Virilizer is the homologous protein in fly, which catalyses m6A formation to control sex determination, and is considered the fifth component of m6A writers (Hilfiker et al ., ; Kan et al ., ). In Arabidopsis , VIRILIZER (KIAA1429 human homolog protein) and the E3 ubiquitin ligase HAKAI (HAKAI human homolog protein) have been found as the fourth and fifth key component, respectively (Bodi et al ., ; Růžička et al ., ).…”