Genes encoding m⁶A writer complexes have been identified and characterized firstly in animals. Several proteins including methyltransferase-like 3 (METTL3) and METTL14, Wilms’ tumor 1-associating protein (WTAP), and Vir like m⁶A methyltransferase-associated (VIRMA; KIAA1429) are known to form multicomponent m⁶A writer complexes in animals (Shah and Clancy, 1992; Ping et al., 2014; Schwartz et al., 2014; Table 1).

Methyltransferase-like 3 is the principal enzyme exerting methyltransferase activity, while METTL14 has a supporting role forming a METTL3-METTL14 heterodimer (Sledz and Jinek, 2016; Wang et al., 2016). After the identification of METTL3 in mammals as a homolog of yeast methyltransferase IME4 (Shah and Clancy, 1992), its orthologs were identified in different species including Arabidopsis and Drosophila. At present, Arabidopsis orthologs of animal m⁶A writer components have been identified, including MTA (ortholog of METTL3) and MTB (ortholog of METTL14).

Wilms’ tumor 1-associating protein functions as a stabilizer for the heterodimer localized in nuclear speckle (Ping et al., 2014; Lence et al., 2016). VIRMA plays a role in guiding the methyltransferase complex to the selective target region of mRNAs (Niessen et al., 2001; Yue et al., 2018). Arabidopsis VIR and FIP37 were identified as a ortholog of VIRMA and WTAP, respectively (Zhong et al., 2008; Bodi et al., 2012; Shen et al., 2016; Růžička et al., 2017).

Recently, zinc finger CCCH domain-containing protein 13 (ZC3H13), the latest component of methyltransferase complex, was found to be essential for localization of methyltransferase complex in mammals and Drosophila (Guo et al., 2018; Knuckles et al., 2018). However, the existence and molecular function of ZC3H13 in plants remain unknown. Interestingly, Arabidopsis contains E3 ubiquitin ligase HAKAI as an additional m⁶A writer component (Růžička et al., 2017; Table 1). Although knockdown of its expression can decrease m⁶A level (Růžička et al., 2017), the primary role of HAKAI in methyltransferase complexes has yet to be investigated.

Removal of methylation marks on RNAs is carried out by α-ketoglutarate-dependent dioxygenase (AlkB) homolog (ALKBH) proteins that can erase alkyl and methyl groups from DNAs, RNAs, and proteins (Fedeles et al., 2015; Alemu et al., 2016). Mammals have nine ALKBH family members: ALKBH1 to ALKBH8 and fat mass- and obesity-associated protein (FTO) (Ougland et al., 2015; Table 1). Although ALKBH2 and ALKBH3 have been identified as main DNA repair enzymes, ALKBH3 also shows activity on m¹A and m³C of RNAs (Ueda et al., 2017). Interestingly, ALKBH1 acts on a wide range of substrates in DNAs, RNAs, and histones (Westbye et al., 2008; Ougland et al., 2012; Wu et al., 2016). In addition to its role in cytoplasm, human ALKBH1 targets several m¹A methylated tRNAs in mitochondria, influencing the organellar translation and function (Kawarada et al., 2017; Müller et al., 2018). ALKBH8, another tRNA DMT, interestingly contains both methyltransferase and demethylase domains, unlike other family members (Pastore et al., 2012).

Only two m⁶A erasers, ALKBH5 and FTO, have been identified in animals so far. Both enzymes were originally shown to be involved in demethylation of m⁶A (Jia et al., 2011; Zheng et al., 2013). However, recent studies have suggested that FTO has a much higher activity toward N⁶, 2^′-O-dimethyladenosine (m⁶A_m) compared to that for m⁶A (Meyer and Jaffrey, 2017; Mauer et al., 2017; Mauer and Jaffrey, 2018). ALKBH5 and FTO have been found to be involved in alternative splicing, 3^′-UTR processing, mRNA stability, translation, and amino-acids deprivation response pathway (Zheng et al., 2013; Zhao et al., 2014; Bartosovic et al., 2017; Tang et al., 2018). Arabidopsis contains several putative m⁶A eraser ALKBH family proteins (Table 1), among which only two eraser proteins ALKBH9B and ALKBH10B have been functionally characterized in viral infection and floral transition (Duan et al., 2017; Martínez-Pérez et al., 2017). In summary, although increasing number of erasers targeting specific methylation marks have been identified, the activity and substrate RNAs of most ALKBH family members in plants and animals are yet to be determined.

Interpretation of methylation marks is tightly related to posttranscriptional regulation of mRNA metabolism which requires reader proteins to recognize methylated transcripts and ultimately determine their fates. In recent years, several RNA-binding proteins (RBPs) that can recognize m⁶A marks on mRNAs have been identified in animals by RNA-protein immunoprecipitation using synthetic m⁶A-containing RNAs (Dominissini et al., 2012; Xu et al., 2014; Arguello et al., 2017; Edupuganti et al., 2017; Wu et al., 2017). YT521-B homology (YTH) domain family (YTHDF) protein was first identified as an m⁶A-binding protein (Xu et al., 2014). Recently, human and mouse YTHDF proteins including YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 were found to possess a specific binding pocket for m⁶A nucleotides and exhibit significantly high affinity to methylated RNAs, suggesting their role as m⁶A readers (Dominissini et al., 2012; Hsu et al., 2017; Xiang et al., 2017; Liao et al., 2018). YHHDF2 can bind to m⁶A-modified RNAs and play a distinct role in mRNA degradation by recruiting the CCR4-NOT deadenylase complex (Wang et al., 2014; Zhou et al., 2015; Du et al., 2016). YTHDF1 was found to recognize the 5^′UTR of m⁶A-modified mRNAs in the cytosol, which promotes translation of target transcripts in a cap-independent manner (Wang et al., 2015b; Shi et al., 2017). YTHDC1 is involved in exon-selective gene splicing in the nucleus (Xiang et al., 2017). Interestingly, YTHDC2 also contains RNA helicase domain (Jain et al., 2018). Arabidopsis and rice genomes encode 13 and 12 YTHD proteins, respectively (Li et al., 2014a; Table 1). Contrary to extensive study on YTHD proteins in animals, only three evolutionarily conserved c-terminal region (ECT) family proteins have recently been functionally characterized in Arabidopsis as YTHD homologs (Arribas-Hernández et al., 2018; Scutenaire et al., 2018; Wei et al., 2018; Table 1).

Besides YTHD proteins, two other proteins containing different RNA-binding domains that can recognize m⁶A marks in animal cells have been reported. One is a heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) which regulate RNA splicing in the nucleus through a well-characterized RNA-recognition motif (Alarcon et al., 2015). Notably, instead of directly binding to m⁶A site as YTHD proteins, HNRNPA2B1 might bind to altered structures right after the m⁶A site (Alarcon et al., 2015). Insulin-like growth factor 2 mRNA-binding protein (IGF2BP) contains tandem K-homology (KH) domains to recognize m⁶A sites and enhance target mRNA stability, storage, and translation in an m⁶A-dependent manner (Nicastro et al., 2015; Huang et al., 2018). Eukaryotic initiation factor 3 (eIF3) can also promote translation of mRNAs depending on m⁶A modification (Meyer et al., 2015). Clearly, more reader proteins recognizing other RNA modifications as well as m⁶A marks should be uncovered to fully understand cellular roles of epitranscriptomic RNA modifications in both plants and animals.

Genome-wide m⁶A methylation patterns have been mapped in barley, Arabidopsis, and rice (Li et al., 2014b; Luo et al., 2014). However, key enzymes responsible for this methylation have only been studied in Arabidopsis. Analysis of mta (Arabidopsis ortholog of human METTL3) knockdown mutants has revealed that MTA is required for m⁶A mRNA methylation and essential for normal growth and development, such as shoot and root growth as well as leaf and floral development (Zhong et al., 2008; Bodi et al., 2012). Moreover, MTA was found to interact with MTB, an Arabidopsis ortholog of human METTLl4. Knockdown of MTB showed a similar but less severe phenotype compared to mta mutants, indicating that both writers are essential for plant development (Růžička et al., 2017). The Arabidopsis m⁶A writer complex also includes an ortholog of human WTAP named FIP37. Depletion of FIP37 results in embryo lethality while its partial loss causes huge overproliferation of shoot meristems by increasing the stability of shootmeristemless (STM) and WUSCHEL (WUS) (Shen et al., 2016). Vir and Hakai are other m⁶A writer components in Arabidopsis. They affects root and shoot growth as well as cotyledon development, similar to other m⁶A writer mutant phenotypes (Růžička et al., 2017). However, the molecular mechanism underlying Vir and Hakai functions is yet to be elucidated.

Despite increasing understanding of the roles of m⁶A writers in plant growth and development, reports demonstrating their involvement and functions in plant response to abiotic stresses are lacking. Our analysis of publically available microarray data using GENEVESTIGATOR revealed that expressions levels of writers in Arabidopsis and rice are differently modulated by diverse abiotic stresses (Figure 3). In Arabidopsis, levels of most m⁶A writer components were not significantly modulated by abiotic stresses. Levels of MTA and FIP37 were only marginally increased by cold and heat stress, respectively. In rice, the level of OsFIP was increased by cold stress whereas levels of OsMTA, OsMTB, and OsVIR were decrease by cold, drought, or salt stress. The constant expression of m⁶A writer components under normal and stress conditions suggests the fundamental role of m⁶A methylation in plant development and stress responses.

Although m⁵C methylation in DNA has been studied for many years, its cellular and molecular functions in RNAs is just beginning to be uncovered. Due to advancement in RNA sequencing, m⁵C RNA methylation could be mapped to mRNAs in both animals and plants (Schaefer et al., 2009; Hussain et al., 2013; Song et al., 2018). Overall, m⁵C RNA methylation is a less abundant modification of mRNA than m⁶A methylation. In Arabidopsis, two enzymes, TRM4A and TRM4B, are responsible for m⁵C RNA methylation. Both enzymes are orthologs of human m⁵C methyltransferase NSuns2. However, TRM4A contributes to tRNA m⁵C methylation while TRM4B targets mRNA for m⁵C methylation. Loss of TRM4A does not exhibit any visible phenotype while loss of Trm4B reduces root length, suggesting the role of mRNA m⁵C methylation in root development regulation (David et al., 2017). In accordance to this, loss of m⁵C RNA methylation affects the stability of short hypocotyl 2 (SHY2) and indoleacetic acid-induced protein 16 (IAA16), two critical genes related to root development (Cui et al., 2017). Our analysis showed that expression levels of Arabidopsis TRM4B are marginally increased by cold stress, although they decrease under heat stress. In contrast, expression levels of rice TRM4A and TRM4B are not altered in response to abiotic stresses (Figure 3). Although these expression patterns suggest potential roles of m⁵C writers in abiotic stress response, the relevance of m⁵C methylation to abiotic stress responses awaits further investigation.

Among protein factors involved in RNA methylation in plants, erasers are so far the least studied, although new knowledge is gained rapidly. Thirteen Arabidopsis ALKBH family members have been identified by bioinformatic analysis (Mielecki et al., 2012). However, only a few of them have been studied so far (Table 1). Among them, ALKBH9A, 9B, 9C, 10A, and 10B show the highest amino acid sequence similarity with human ALKBH5. Other family members are numbered based on their sequence similarity to human orthologs (Table 1). Like animal counterparts, most erasers are localized in the nucleus and cytoplasm whereas ALKBH1D is also present in chloroplasts. Interestingly, some of them show relocalization to the nucleus in response to methylating agents (Mielecki et al., 2012). ALKBH10B was identified as the principal mRNA m⁶A eraser influencing floral transition by controlling transcript levels of SPL3, SPL9, and FLOWERING LOCUS T (Duan et al., 2017). Another demethylase, ALKBH9B, was shown to revert m⁶A from single-stranded RNA in vitro (Martínez-Pérez et al., 2017). Although alkbh9b knockout mutants do not show differences in plant RNA m⁶A methylation level (Duan et al., 2017), its depletion results in hypermethylation of alfalfa mosaic virus (AMV) RNA, mediating systemic infection by interacting with viral cap proteins (Martínez-Pérez et al., 2017).

Expression of ALKBH9A is highly induced in roots under salt stress but not in response to ABA (Ma et al., 2006). Its level is much lower than ALKBH9 and ALKBH10 under normal conditions (Duan et al., 2017). ALKBH10A is down-regulated by heat stress (Merret et al., 2015) whereas ALKBH10B is up-regulated in response to karrikins (Nelson et al., 2010). Although these previous studies suggest a specific role of ALKBHs in stress responses as well as plant development, nothing is known about their actual roles. Our analysis showed that expression levels of ALKBH members were marginally up- or down-regulated in Arabidopsis by different abiotic stresses (Figure 3). Notably, levels of ALKBH1 in rice were highly increased upon drought, cold, or ABA treatment whereas expression levels of ALKBH6, ALKBH8B, and ALKBH10A were decreased by drought, ABA, or cold (Figure 3). These data suggest that ALKBHs could play important roles in abiotic stress responses, although this awaits further investigation.

Although several RBPs interpreting m⁶A marks have been identified in animals, roles of only three YTHD m⁶A reader proteins have very recently been determined in Arabidopsis (Arribas-Hernández et al., 2018; Scutenaire et al., 2018; Wei et al., 2018). YTHD09 (ECT2) is involved in trichome development. Moreover, cytoplasmic-localized YTHD09 relocates to stress granules upon heat exposure, suggesting its role in mRNA fate control under stress conditions (Scutenaire et al., 2018). By using single and double mutants, it has been demonstrated that YTHD09 (ECT2), YTHD13 (ECT3), and ECT4 regulate the timing and execution of plant organogenesis (Arribas-Hernández et al., 2018). Moreover, a molecular study revealed that ECT2 targets a large number of m⁶A-containing transcripts, including TTG1, ITB1, and DIS2, which are involved in trichome development (Wei et al., 2018). Further sequencing analysis suggested that ECT2 increases the stability of these transcripts and influences trichome development (Wei et al., 2018). Although these studies clearly point to important roles of YTHD readers in plant development, more in-depth and focused efforts are needed to identify and characterize potential reader proteins (Table 1) that can recognize not only m⁶A modification, but also other methylation marks in plants.

No reports demonstrating the involvement or functions of any RNA methylation reader proteins in plant response to abiotic stresses have been published so far. However, a previous study and our current analysis showed that the expression of YTHDs in Arabidopsis and rice is highly regulated by different abiotic stresses (Li et al., 2014a; Figure 3). In Arabidopsis, levels of YTHD05, YTHD06, and YTHD07 are increased by heat, cold, hypoxia, or submergence stress. In contrast, the expression level of YTHD10 decreases under cold, drought, salt, or osmotic stress whereas YTHD08 level is reduced by heat stress. In rice, YTHDs responded differently to various abiotic stresses (Li et al., 2014a; Figure 3). Expression levels of YTHD05, YTHD06, YTHD07, and YTHD09 are downregulated by cold stress whereas levels of YTHD03 and YTHD08 increase under submergence and heat stress, respectively. Notably, none of these rice YTHDs showed altered expression under salt stress whereas YTHD01, YTHD02, YTHD03, YTHD04, or YTHD08 does not respond to cold stress. The fact that m⁶A reader proteins respond more to abiotic stresses than writers and erasers suggests that decoding of methylation marks is much more important than introducing or removing these marks during stress adaptation process in plants. It would be interesting to characterize roles of reader proteins in RNA metabolism and its consequence in stress responses.