MTA Is an Arabidopsis Messenger RNA Adenosine Methylase and Interacts with a Homolog of a Sex-Specific Splicing Factor  

Abstract

N 6 -Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N 6 -methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N 6 -methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N 6 -methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

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

“…We used tandem affinity purification (TAP) followed by proteolysis and mass spectrometry (Van Leene et al ., 2011) to identify proteins that associated with the VIR‐GS bait in Arabidopsis suspension cell cultures. Using this approach, we consistently identified FIP37 (Zhong et al ., 2008) and HAKAI (Fig. S3; Fujita et al ., 2002; Horiuchi et al ., 2013) co‐purifying with VIR and MTB (Bujnicki et al ., 2002) also significantly enriched with a lower confidence score (Fig.…”

“…The quantification of relative m 6 A levels was performed as described previously (Zhong et al ., 2008). Briefly, 20 μg of total RNA was extracted from Arabidopsis seedling samples (or from roots of β‐estradiol treated inducible RNAi lines) using the RNAqueous kit (Ambion), the poly(A) + fraction was purified twice using the MicroPoly(A) Purist kit (Ambion) and the quality of the mRNA checked on an RNA 6000 LabChip, with an Agilent Bioanalyzer (Ambion).…”

“…The protein–protein interactions were tested as described previously (Zhong et al ., 2008; Pekárová et al ., 2011). Plasmids were constructed by Gateway‐based technology using destination vectors pDEST22 (activation domain) and pDEST32 (binding domain).…”

“…m 6 A appears to be involved in a broad range of biological processes including mRNA export from the nucleus (Fustin et al ., 2013), regulation of splicing (Alarcón et al ., 2015b; Haussmann et al ., 2016; Lence et al ., 2016), mRNA translatability and stability (Wang et al ., 2014a,b, 2015; Bodi et al ., 2015; Zhou et al ., 2015), alternative polyadenylation site choice (Ke et al ., 2015) and other mechanisms accompanying RNA maturation (Meyer & Jaffrey, 2014; Yue et al ., 2015). m 6 A is essential for the earliest stages of pattern formation in plants (Zhong et al ., 2008; Bodi et al ., 2012; Shen et al ., 2016) and metazoans (Meyer & Jaffrey, 2014; Geula et al ., 2015; Yue et al ., 2015; Haussmann et al ., 2016; Lence et al ., 2016), linked with diseases in humans and other mammalian species (Jia et al ., 2011; Zheng et al ., 2013) and is required for meiosis in Saccharomyces cerevisiae (Clancy et al ., 2002). Reduced levels of m 6 A also affect circadian period (Fustin et al ., 2013) and are critical for stem cell differentiation in mammals (Geula et al ., 2015).…”

“…Whilst METTL3 was part of a 200‐kDa complex, a further 875‐kDa complex component(s) was required for its in vitro activity (Bokar et al ., 1994, 1997). The identity of any of the other proteins that act together with METTL3 remained unknown until it was shown in Arabidopsis that FIP37 (FKBP12 INTERACTING PROTEIN 37) was a partner protein of MTA (the homologue of METTL3) (Zhong et al ., 2008; Shen et al ., 2016). Following this initial discovery, the homologues of FIP37, S. cerevisiae MUM2 (Muddled Meiosis 2), and mammalian WTAP (Wilms tumour 1 associated protein), were shown to interact with METTL3 (MTA) orthologues and to be required for mRNA methylation in their respective model organisms (Agarwala et al ., 2012; Ping et al ., 2014; Wang et al ., 2014b; Liu et al ., 2015).…”