Dynamic N6-methyladenosine RNA methylation in brain and diseases

Abstract

N6-methyladenosine (m⁶A) is a dynamic RNA modification that regulates various aspects of RNA metabolism and has been implicated in many biological processes and transitions. m⁶A is highly abundant in the brain; however, only recently has the role of m⁶A in brain development been a focus. The machinery that controls m⁶A is critically important for proper neurodevelopment, and the precise mechanisms by which m⁶A regulates these processes are starting to emerge. However, the role of m⁶A in neurodegenerative and neuropsychiatric diseases still requires much elucidation. This review discusses and summarizes the current body of knowledge surrounding the function of the m⁶A modification in regulating normal brain development, neurodegenerative diseases and outlines possible future directions.

Tight regulation of the nervous system is required to ensure proper function, and predictably, anomalies during brain development have been associated with neurological disorders. Indeed, several neural and non-neural cell types must be generated at the required numbers, in the appropriate location, at a specific time for proper brain development. Furthermore, accurate synaptic connections between neurons and efficient communication between distinct cell populations are essential for the proper function of the brain. Indeed, many studies have provided a greater understanding into human neurodevelopmental processes [1–5]. Clearly, such an orchestrated, precise developmental plan requires a multilayered genetic blueprint to maintain proper development. To establish the correct RNAs and proteins at each stage of neurogenesis, dynamic, rapid changes in gene expression are required. To accomplish this there needs to be precise, time-sensitive mechanisms in place in order to maintain proper brain development. Both epigenetic and transcriptional mechanisms have been shown to control neurogenesis. For example, DNA methylation and histone modification-mediated gene regulation are crucial for neural cell differentiation [6–9]. Furthermore, transcriptional regulation has also been shown to have a vital role in regulating neurogenesis [10–12]. Transcriptional control is exerted by transcription factors that regulate processes such as cell cycle exit, loss of progenitor properties and acquisition of neuronal features [13–17]. In addition to epigenetic and transcriptional control, epitranscriptomic regulation could potentially afford yet another layer of regulation.

The most abundant epitranscriptomic mark is the N6-methyladenosine (m⁶A) modification. This modification is dynamic and is deposited onto mRNA by ‘writer’ methyltransferases (METTL3/METTL14) [18–20] in complex with the METTL3 adaptor, WTAP [21], and other associated proteins, KIAA1429 [22] and RBM15/15B [23]. ‘Eraser’ enzymes (FTO, ALKBH5) remove the modification [24,25], and its function is mediated by proteins that specifically recognize it, ‘reader’ proteins (e.g., YTHDF 1, 2, 3) [26–28]. m⁶A has been implicated in mRNA stability, translation, splicing and miRNA processing [27–31]. Depending on the specific context, specific reader proteins will recognize the m⁶A mark and mediate its function (for a more detailed review see Shi H, Wei J, He C [32]; Figure 1). Furthermore, as m⁶A is a dynamic modification, mRNAs can be rapidly marked and unmarked, allowing for tight, fast regulation of mRNAs. Altogether, it is conceivable that m⁶A could exert regulatory control over a biological process. This review focuses on the role of m⁶A in neurodevelopmental pathways (see Table 1 for a summary) and neurodegenerative disease (see Table 2 for a summary).

Figure 1. . N6-methyladenosine affects many aspects of RNA metabolism.

(A) mRNA stability – YTHDF2 specifically reads m⁶A marked transcripts localizing the mRNA to P-bodies for degradation. (B) Translation – YTHDF1 and 3 specifically recognize m⁶A marked transcripts, resulting in enhanced translation through a closed-loop model. (C) Splicing – YTHDC1 is a m⁶A reader which directs the splicing factor SRSF3 to its target, resulting in exon inclusion. (D) miRNA processing – HNRNPA2/B1, in complex with DGCR8, recognizes primary miRNAs modified by m⁶A delivering DGCR8 to its target for enhanced miRNA processing.

m⁶A: N6-methyladenosine.

Table 1. . Role of N6-methyladenosine in neurodevelopment.

Developmental pathway	Related enzymes and proteins	Role of m6A	Key publications
Cerebellar development	METTL3: KO negatively affects Purkinje cell numbers, laminal structure, dendrite formation and the organization of glial cell fibers	m6A both positively and negatively modulates gene expression of genes important in proper cerebellar development	Ma et al. (2018) [36] Chang et al. (2017) [37]
Neurogenesis	METTL14: KO results in a reduction in cortical length and thickness FMRP: KO results extended neuronal progenitor cell cycle FTO: KO leads to decreased brain size and body weight YTHDF2: KO has a strong negative impact on NSPC self-renewal and neuron generation in embryonic neocortex	m6A marks transcripts for degradation to ensure proper cortical neurogenesis m6A is involved in transcriptional pre-patterning for cortical neurogenesis m6A could regulate the BDNF pathway, and this may be linked to postnatal neurogenesis	Yoon et al. (2017) [35] Wang et al. (2018) [43] Edens et al. (2019) [44] Zhang et al. (2018) [45] Li, Weng et al. (2017) [46] Li, Zhao et al. (2018) [47]
Synaptogenesis	YTHDF1 and 3: KO results in altered spine morphology, dampened excitatory synaptic transmission, and altered cell-surface protein content	m6A marked mRNAs known to function in synaptic plasticity	Merkurjev et al. (2018) [48] Koranda et al. (2018) [49]

m⁶A: N6-methyladenosine; NSPC: Neural stem/progenitor cell.

Table 2. . Role of N6-methyladenosine and components of N6-methyladenosine pathways in neurodegenerative and neuropsychiatric diseases.

Disease	Related enzyme and proteins	Role of m6A	Key publications
Deficit hyperactivity disorder in children, major depressive disorder and Alzheimer’s disease	FTO	No known role	Choudhry et al. (2013) [52] Velders et al. (2012) [53] Milaneschi et al. (2014) [54] Keller et al. (2011) [56] Reitz et al. (2012) [57]
Parkinson’s disease	FTO	m6A may affect dopaminergic signaling	Hess et al. (2013) [55] Chen et al. (2019) [62]
Autism spectrum disorder	YTHDC2	No known role	Liu et al. (2016) [58]
Stress-related psychiatric disorders	FTO ? Conflicting data as to whether FTO has a positive or negative affect on anxiety and depression	Altered levels of m6A was observed in depressed patients after glucocorticoid stimulation	Engel et al. (2018) [59] Spychala, Ruther (2019) [60] Sun et al. (2019) [61]

m⁶A: N6-methyladenosine.

m⁶A in brain development

Both gene expression and environment are critical for normal brain development, and clearly, perturbation in either can negatively affect neural outcomes. Brain development is correctly maintained through context-specific dynamic and adaptive processes that are perfectly regulated for proper neural structure and functions [33]. With the dynamic nature of m⁶A and its multifaceted functionality, m⁶A adds another layer of regulation during brain development. m⁶A has been shown to be highly pervasive in the brain, more so in humans compared with mice, and, there is a high level of conservation between the two, suggesting a pivotal role for m⁶A in brain development [34,35].

Two recent studies have focused on understanding the postnatal m⁶A landscape in the mouse brain. Ma C, Chang M, Lv H et al. [36] profiled m⁶A in the mouse cerebellum across four postnatal developmental stages ranging from 7 days to 60 days postbirth (P7, P14, P21 and P60). Chang M, Lv H, Zhang W et al. [37] have provided m⁶A maps in both the cerebellum and cortex of 2-month old mice. In the first study, they identified a similar number of methylated mRNAs across the four stages and similar methylation patterns across all four stages. Interestingly, they observed a significant number of m⁶A peaks in start codons, which increased slightly from P7 to P60. A considerable number of m⁶A peaks were also identified in the coding region and stop codon. To glean insight into m⁶A function, they identified differential m⁶A peaks in a pairwise fashion. They noted the presence of 12,452 emerging m⁶A peaks or ‘ON’ peaks (i.e., peaks present only at a later stage, when compared with an earlier stage), and 11,192 disappearing m⁶A peaks or ‘OFF’ peaks (i.e., peaks present only at an earlier stage, when compared with a later stage). The distribution of these m⁶A peaks varied by type: ‘ON’ switches were more likely to be deposited at start codons, whereas ‘OFF’ switches were more observed around stop codons. Importantly, the authors find that the majority of m⁶A peaks are present at all time points, however, they were also able to identify some temporal specific peaks. Chang M, Lv H, Zhang W et al. [37] also identified common and region specific m⁶A methylation. The common m⁶A peaks were typically located at the stop codons and 3′ UTR. However, cerebellum-specific peaks were distributed in the start codons and 3′ UTR, while cortex-specific peaks were mainly concentrated in the coding region. Ma C, Chang M, Lv H et al. [36] reported similar findings. They noted that the distribution of P7-specific m⁶A peaks were enriched at the stop codon, while P60-specific m⁶A peaks were enriched at the start codon. Gene ontology analyses revealed that biological processes such as cell cycle, cell division and DNA repair were strong for genes containing P7–P14 OFF switches. However, genes that contain ‘ON’ methylation switches are involved in signal transduction, cell adhesion, learning and synaptic plasticity. Furthermore, they show that the m⁶A methylation is able to positively and negatively affect gene expression. Thus, m⁶A is clearly important for cerebellar development by modulating gene expression of cell-fate determining genes. In the same study, localization studies of the five major players in the m⁶A pathway (METTL3, METTL14, WTAP, ALKBH5 and FTO) revealed that all five proteins were present in the external granule cell layer, Purkinje cell layer and the inner granule cell layer [36]. Knockdown of Mettl3 was shown to negatively affect Purkinje cell numbers, laminal structure, dendrite formation and the organization of glial cell fibers [36]. Interestingly, expression levels of the five proteins decreased when transitioning from P7 to P60, suggesting that m⁶A has a more crucial role early in cerebellar development. Chang M, Lv H, Zhang W et al. [37] also found that protein levels of the m⁶A machinery are higher in the cerebellum than in the cortex. Taken together, these findings suggest that m⁶A is a significant regulator of developmental processes in cerebellum and cortex.

m⁶A in neurogenesis

During development, neurogenesis results in the large diversity of neurons in the brain. During the process, neural stem cells (NSCs) differentiate at specific times and regions in the brain. In neurogenesis, stem cells have the capacity for self-renewal through cell division, or they can differentiate into specialized cell types, such as neural progenitor cells [38–40]. These progenitor cells themselves then can differentiate into specific types of neurons. Clearly, proper maintenance of neurogenesis is essential for normal neurodevelopment, and perturbation of the NSC self-renewal and neurogenesis at any time during development can directly lead to neurological and psychiatric disorders [41,42]. Two recent papers have demonstrated that m⁶A exerts some control over the neurogenesis process.

To study the effect of METTL14-mediated m⁶A on neurogenesis, two prominent studies employed the use of a conditional Mettl14 KO x Nestin-cre mouse model [35,43]. Yoon KJ, Ringeling FR, Vissers C et al. [35] showed that the conditional Mettl14 KO (cKO) in mice produced animals that were visibly smaller in size at P5 compared with littermate controls, and those cKO animals failed to survive past P25. Comparison of the Mettl14 cKO to the WT revealed differences in cortical structure and development. Wang Y, Li Y, Yue M et al. [43] reported that the cKO pups were viable, did not display any obvious morphologic phenotypes and appeared to have normal body weight at P0. However, all Mettl14-cKO mice were dead within the first neonatal week. Wang Y, Li Y, Yue M et al. [43] also observed a reduction in cortical length and thickness, and both studies found an enlargement of the ventricle. Altogether, these results demonstrate a critical function for METTL14 in neurogenesis. Furthermore, Yoon KJ, Ringeling FR, Vissers C et al. [35], observed METTL14-induced m⁶A depletion resulted in prolonged cell cycle of adult radial glial, whereas neuron production still occurred into postnatal stages: an observation clearly implicating m⁶A as a significant regulator of cortical neurogenesis [35]. Wang Y, Li Y, Yue M et al. [43] also reported that the Mettl14 cKO resulted in a loss of late neurons, further suggesting a role for METTL14 in cortical neurogenesis. These in vivo findings were further bolstered by performing m⁶A-seq on E13.5 mouse forebrains (where NSCs are prominent) [35]. These authors found that many transcripts that encode transcription factors, such as Pax6, Sox1, Sox2, Emx2 and Neurog2/Neurogenin 2, are marked with m⁶A. A gene ontology analysis of m⁶A marked transcripts yielded enrichment in cell cycle, stem cell and neuronal differentiation pathways. m⁶A in this context, marks the transcripts for degradation, maintaining proper cortical neurogenesis. Aberrant m⁶A during the process negatively affects temporal specification and cell cycle progression of neuronal progenitor cells [35]. Further, a recent study uncovered an additional layer of regulation for transcripts involved in neurogenesis. Edens BM, Vissers C, Su J et al. [44] showed that, during neural differentiation, fragile X mental retardation protein (FMRP) can specifically read the m⁶A modification facilitating the nuclear export of m⁶A-marked transcripts (Figure 2A). Zhang F, Kang Y, Wang M et al. [45] also showed that mRNA targets of FMRP are enriched for m⁶A marks. Similar to what was described in the Mettl14 cKO mice, Fmr1 KO mice exhibited an extended neuronal progenitor cell cycle, with neural progenitors still proliferating into postnatal stages. From these studies, it is clear that both FMRP and METTL14 are required for neural progenitor differentiation (Figure 2A & B). At last, transcriptome-wide m⁶A profiling also showed that m⁶A has a role in actively suppressing transcription of mRNAs present in adult radial glial, but whose protein product is only functional downstream in the lineage, suggesting that m⁶A is involved in transcriptional pre-patterning for cortical neurogenesis [35]. The advantage of such an epitranscriptomic post-transcriptional mechanism is that the dynamic m⁶A mark can suppress translation (e.g., by marking the transcript for decay), but then can be removed rapidly to allow translation to occur whenever the protein is required.

Figure 2. . N6-methyladenosine is critically important in the brain.

(A) FMRP facilitates nuclear export of m⁶A marked transcripts. (B) m⁶A promotes decay of these transcripts to maintain proper cortical neurogenesis. (C) YTHDF1 and 3 are required to recognize m⁶A at synapses to ensure proper synaptic function. (D) m⁶A results in the decay of the CBP/p300 acetyltransferase mRNA, which in turn results in decreased histone modifications, ultimately regulating gene expression.

m⁶A: N6-methyladenosine.

A study by Li Z, Weng H, Su R et al. [46] provided evidence that FTO-catalyzed m⁶A modification is important in neurogenesis, learning and memory. They found FTO in both adult NSCs and neurons. They further showed an increase in expression of FTO from postnatal day 1 to postnatal 8 weeks. To characterize the role of m⁶A regulated by FTO, the authors generated an m⁶A map at 2- and 6-week-old, and compared that to RNA-seq data from wild-type and Fto KO mice. They found 363 genes that had altered expression were also marked with m⁶A. These genes were associated with neuronal development, cell proliferation and migration pathways. Furthermore, they note that brain-derived neurotrophic factor (BDNF) is important for neurogenesis and is regulated by FTO. They found that many players in the BDNF pathway are m⁶A methylated at both 2 and 6 weeks, and have reduced expression in the Fto KO mice compared with WT. These results suggest that FTO-catalyzed m⁶A modification could regulate the BDNF pathway, and, in turn, this may affect postnatal neurogenesis. Another study by Li M, Zhao X, Wang W et al. [47] further implicated m⁶A in neurogenesis by looking at Ythdf2 knockdown mice. They showed that YTHDF2 is highly expressed during the early stage of neural development and Ythdf2^−/− mouse embryos at E12.5 and E14.5 were alive but displayed reduced overall cortical thickness, as is seen in Mettl14 cKO mice [47]. In addition, they showed that loss of Ythdf2 has a strong negative impact on neural stem/progenitor cell self-renewal and neuron generation in embryonic neocortex. To gain insight into the molecular mechanism, the authors performed RNA-seq and compared gene expression in Ythdf2^−/− and wild-type mice. They found that differentially expressed genes had functions related to axon regulation, synapse assembly and neuron differentiation. Interestingly, these genes, such as, Ddr2, Rnf135, Flrt2, Hlf, Nrp2, Nrxn3 and Ptprd have a marked increase in expression and m⁶A levels in Ythdf2^−/− compared with WT. This study provides evidence that YTHDF2-mediated m⁶A plays an important role in neurogenesis during embryonic neural development. Critically, m⁶A has also been implicated as a major regulator of synaptic function. Merkurjev D, Hong WT, Iida K et al. [48] profiled m⁶A modifications of synaptosomal RNA and discovered 2921 methylated genes comprising the ‘synaptic m⁶A epitranscriptome’. This epitranscriptome is important in many pathways involved in neurodevelopment and neuropsychiatric diseases. Indeed, they reported that the m⁶A reader proteins YTHDF1, YTHDF2 and YTHDF3 are enriched in hippocampal dendrites, and the loss of YTHDF1 or YTHDF3 there resulted in altered spine morphology, dampened excitatory synaptic transmission and altered cell-surface protein content (Figure 2C) [48]. Furthermore, mRNAs that are specifically associated with synaptic function were both enriched and differentially methylated in synapses. Altogether, these observations provide evidence that m⁶A at synapses is required for proper synaptic function. Koranda JL, Dore L, Shi H et al. [49] also suggested a role for m⁶A in synaptic signaling. They profiled m⁶A in Mettl14-deleted striatum where they found a correlation between loss of m⁶A and downregulation of those mRNAs. Interestingly, gene ontology analysis showed that these downregulated mRNAs encode neuron and synapse-specific proteins. More specifically, they found m⁶A methylation in mRNAs known to function in synaptic plasticity, such as Homer1 and Cdk5r1.

Link between m⁶A, neurogenesis & histone modifications

By deleting Mettl14 in embryonic NSCs, Wang Y, Li Y, Yue M et al. [43] observed a significant reduction in proliferation, which in turn resulted in their premature differentiation. These findings suggest METTL14 regulates NSC self-renewal. An RNA-seq analysis revealed Mettl14 cKO NSCs displayed an altered gene expression profile compared with the control. Significantly upregulated genes are involved in NSC differentiation, whereas significantly downregulated genes are associated with cell proliferation; the observed phenotype of these cells concurred with the changes in gene expression. The authors only observed a mild correlation between m⁶A and mRNA levels, suggesting another mechanism was at play here. Interestingly, they observed significantly increased levels of histone modifications (e.g., histone H3 at lysine 27, trimethylation of histone H3 at lysine 4 and trimethylation of histone H3 at lysine 27) in Mettl14 KO NSCs compared with control. Furthermore, by using a chemical inhibitor to block H3K27me3 and H3K27ac, the authors showed that the observed abnormalities of Mettl14 KO NSCs could be rescued. At last, they detected m⁶A methylation in transcripts that encoded the H3K27 acetyltransferases CBP and p300 in the control sample but the m⁶A methylation was lost in Mettl14 KO NSCs. Methylation in these two transcripts was directly correlated with mRNA abundance. These findings implicate METTL14 (and m⁶A) as a specific regulator of histone modifiers, which in turn regulates gene expression (Figure 2D). Chen J, Zhang YC, Huang C et al. [50] also reported similar findings: they showed that m⁶A is present on the transcript of the histone methyltransferase Ezh2. Loss of Mettl3 resulted in a reduction of EZH2 protein levels and a concomitant decrease in H3K27me3. They also characterized the effect of METTL3 on neurogenesis. Knockdown of Mettl3 resulted in reduced m⁶A levels in adult NSCs, manifesting itself in the reduced proliferation of adult NSCs, and, furthermore, directed their differentiation toward the glial lineage. Also, the maturation of newborn neurons was affected upon knockdown of Mettl3. Chen J, Zhang YC, Huang C et al. [50] showed that overexpression of Ezh2 could rescue the defects of neurogenesis and neuronal development that were observed following the loss of Mettl3.

m⁶A in neurodegenerative & neuropsychiatric disease

Currently, there is little known regarding the specific role, if any, of m⁶A in neurodegenerative and neuropsychiatric diseases. However, there is strong evidence to suggest a fundamental role for m⁶A in these diseases. The current knowledge of the role of m⁶A players and m⁶A in neurodegenerative and neuropsychiatric disease is summarized in Table 2. As discussed previously, m⁶A is present in synapses, and aberrant translation at synapses has been associated with autism, fragile X syndrome and other intellectual disorders, suggesting there may be a role for m⁶A in these diseases. In addition, m⁶A machinery, but not m⁶A itself, has been implicated in some neurodegenerative diseases. For example, single nucleotide polymorphisms in FTO have been implicated in many neuropsychiatric diseases [51]. The variants have been linked to attention deficit hyperactivity disorder in children [52,53], major depressive disorder [54] and Alzheimer’s disease [55–57]. Also, mutations in YTHDC2 have been implicated as a risk factor for autism spectrum disorder [58]. Engel M, Eggert C, Kaplick PM et al. [59] found that depressed patients have altered levels of m⁶A and m⁶Am (N⁶, 2’-O-dimethyladenosine) after glucocorticoid stimulation, suggesting that these modifications may also have a role in stress-related psychiatric disorders. The same study also reported no change in anxiety levels were observed in Mettl3 or Fto conditional knockout mice. However, Spychala A, Ruther U [60] did see an increase in anxiety following Fto knockout; whereas, Sun L, Ma L, Zhang H et al. [61] reported that FTO deficiency reduced anxiety and depression-like behaviors. FTO, and indeed m⁶A, may also be associated with Parkinson’s disease (PD). Hess ME, Hess S, Meyer KD et al. [55] showed that dopaminergic signaling is negatively affected upon the inactivation of FTO. More recently, Chen X, Yu C, Guo M et al. [62] looked more closely into the role of m⁶A in PD. They modeled the disease in rats and PC12 cells by employing 6-OHDA to selectively destroy dopaminergic neurons. Upon this treatment they found that the m⁶A modification was reduced in the PC12 cells and in the striatum of the PD rat but not the cortex, hippocampus or midbrain. The authors hypothesized that loss of m⁶A in the striatum may result from the increased levels of FTO in the midbrain being transmitted to the striatum via the axons of dopaminergic neurons. There are many potential links between m⁶A, neurodegenerative and neuropsychiatric disease, which may result in attractive targets for therapeutic intervention strategies in patients. For example, the key regulators of the m⁶A modification (especially the writers and erasers) may act as potential therapeutic targets. Clearly, though, more work is needed to uncover the exact mechanisms by which m⁶A may affect these diseases, and how this information can be utilized for therapeutics.

Conclusion & future perspective

m⁶A mRNA methylation is clearly a critical regulator of gene expression in the developing mammalian brain. Further work is needed to fully elucidate the role of m⁶A in brain development and disease. For example, what is the biological significance of the location of m⁶A within the mRNA in the context of neural development and neurological disease? How does the position of the m⁶A affect RNA binding protein binding and how does this contribute to development and disease? This is pertinent because anomalies in RNA processing can result in neurological disease. It is important to understand how m⁶A affects the RNA binding protein landscape. Also, what cues are required to control the level of m⁶A, and how does the mark specifically mediate its function in this context? Mechanistically, more work is required to fully appreciate the nuances of regulation by m⁶A. Emerging long read sequencing technologies such as Nanopore and PacBio will provide us with insights into whether m⁶A has a preference for a specific mRNA isoform or not. Direct RNA sequencing may also shed light on the different epitranscriptomic marks present on specific mRNAs at a given time. This will indicate if modifications work in concert, starting to provide us with a more comprehensive view of how the epitranscriptome functions. Also, with the advent of single-cell sequencing, studies have started to emerge outlining the view of the transcriptome in each cell type in the brain; this will no doubt be extended to the epitranscriptome as well. This will broaden our understanding of differences in m⁶A in different neural cell types and the specific impact of m⁶A on the specific development of particular neural types. Certainly, m⁶A has an important role in maintaining and modulating multiple processes and pathways in the human brain, the details of which still need to be elucidated. Indeed, a strong emphasis should be placed for determining the role of m⁶A in the brain.

Executive summary.

• N6-methyladenosine (m⁶A) is known to affect many aspects of RNA metabolism including mRNA stability, translation, splicing and miRNA processing.

• m⁶A is a dynamic modification and is critical in regulating brain development by both positively and negatively modulating gene expression in genes important for proper development.

• During brain development, distribution of m⁶A along genes is dependent on both spatial and temporal cues, with different m⁶A localization along the gene having a different functional outcome.

• m⁶A marks transcripts for degradation to maintain proper cortical neurogenesis. Aberrant m⁶A results in problems with temporal affects temporal specification and cell cycle progression of neuronal progenitor cells.

• The m⁶A demethylase, FTO, is important in neurogenesis, learning and memory.

• YTHDF2-mediated m⁶A plays an important role in neurogenesis during embryonic neural development.

• m⁶A methylation is present in mRNAs known to function in synaptic plasticity, such as Homer1 and Cdk5r1.

• Aberrant translation at synapses has been associated with autism, fragile X syndrome and other intellectual disorders, thus it is possible that m⁶A may be play a role in those diseases.