N6-methyladenosine (m6A) abundantly exists in the cerebral cortex and is emerging as an essential factor in cortical development and function. As the m6A-binding site appears to be dynamically methylated in different RNA regions at the temporal-specific developing stage, it is of value to distinguish the unique character of region- and temporal-specific m6A. Herein, we analyzed the status of temporal-specific m6A within RNA 5′ untranslated region (5′UTR) using m6A-methylated sequencing data and transcriptomic sequencing data from 12.5- to 13-day embryonic cerebral cortices and 14-day postnatal ones. We identified sorts of RNAs that are uniquely m6A-methylated in the 5′UTR and sorted them into specific neurological processes. Compared with 3′UTR-m6A-methylated RNAs, 5′UTR-m6A-methylated RNAs showed unique functions and mechanisms in regulating cortical development, especially through the pathway of mRNA transport and surveillance. Moreover, the 5′UTR-specific m6A was associated with neurological disorders as well. The FoxO signaling pathway was then focused by these pathogenic 5′UTR-m6A-methylated RNAs and explored to be involved in the determination of neurological disorders. Additionally, the 5′UTR-m6A modification patterns and transcriptional patterns play independent but cohesive roles in the developing cortices. Our study emphasizes the importance of 5′UTR-specific m6A in the developing cortex and provides an informative reference for future studies of 5′UTR-specific m6A in normal cortical development and neurological disorders.

In the last few decades, epigenetics has got more and more attention and displays essential roles in phenotype expression [1]. Plenty of epigenetic-modification patterns were discovered and studied, including RNA methylation. Most recently, N6-methyladenosine (m6A) has been revealed to be the most prevalent and reversible RNA internal modification. As m6A affects mRNA processing and metabolism [2‒5], it plays a crucial role in controlling varied mRNA-related cellular processes. Tissue development, in particular, is spatially and temporally governed by the dynamic m6A modification [6, 7]. Dysregulation of m6A modification is demonstrated to be a typical signature of tissue disorder [8, 9]. Considering the fact that RNA m6A modification abundantly exists in the central nervous system [10, 11], neurologists have been inspired to focus on the regulatory role of m6A modification in brain development and function.

In mammals, the dynamic RNA m6A modification is generally “written,” “read,” and “erased” by methyltransferases (METTL3, METTL14, and WTAP), accessory factors (YTHDF and HNRNPC), and demethylases (FTO and ALKBH5) [12‒14], respectively. Aberrance of these proteins resulted in substantial changes of m6A modification and thus led to the changes in neural development [15]. For instance, Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages [16]. Conditional depletion of the m6A reader (Ythdf2) caused severe abnormalities in the self-renewal of neural progenitor cell and neuron generation in the mouse embryonic neocortex [17]. Alkbh5 deletion disturbed the cell proliferation and differentiation in the mouse cerebellum through disarranging the m6A modification of related genes [18, 19]. Moreover, loss-of-function m6A demethylase (FTO) blocks the postnatal growth of both mice and humans and leads to microcephaly [20, 21]. Globally elevation of m6A methylation degree promoted the development of Alzheimer’s disease [22], whereas downregulation of global m6A modification was found in the brain of rats with Parkinson’s disease [23]. m6A also responses to injury reaction and axon regeneration in the adult mouse brain [24]. These studies further uncover the effects of m6A modification in both brain development and disorders. Nevertheless, the dynamic m6A-modified patterns, as well as its molecular mechanisms, remain misty in brain development and function.

Recently, a powerful technique, m6A-modified RNA immunoprecipitation sequencing (m6A-RIP-seq), has been widely used to profile RNA m6A modification landscape [25, 26]. The presence of m6A-RIP-seq boosted the studies of m6A modification patterns and functions in the brain. As a consequence, a low level of m6A modification was observed throughout embryogenesis, whereas it sharply increased in the adulthood rat brain [27]. In the postnatal mouse cerebellum, m6A modification was temporally switched “ON” or “OFF” to regulate gene expression and thus diversify neural processes, such as proliferation and maturation [19]. Moreover, m6A-RIP-seq also allowed these studies to investigate the features of m6A distribution, which showed an obvious preference in the 3′-untranslated region (3′UTR) and near stop codon (NSC) rather than the 5′ untranslated region (5′UTR) [27]. Subsequently, the functions of 3′UTR-specific m6A were intensively studied and proved to be correlated with alternative polyadenylation of transcripts [28, 29]. In the developing cerebral cortex, the 3′UTR- or NSC-specific m6A was focused and revealed to be involved in cortical morphogenesis and neurological disorders by regulating related gene expression [30]. 5′UTR-specific m6A however achieves less attention but burdens the roles as essential as 3′UTR-specific m6A. Although recent studies have demonstrated that 5′UTR-specific m6A can respond to a variety of cellular stresses [31, 32], more analysis is needed to comprehensively understand the function of 5′UTR-specific m6A, especially in the developing cerebral cortex.

Herein, we deeply explore both m6A-RIP-seq data and RNA transcriptomic data of developing mouse cerebral cortices. We uniquely analyzed the profile of mRNA 5′UTR-specific m6A and its temporal dynamics in mouse cerebral cortices from the embryonic stage to postnatal stage. As compared to 3′UTR-specific m6A, we further identify a distinct role of 5′UTR-specific m6A in the developing mouse cerebral cortex. Although further experimental evidence is needed, our findings provided a better understanding of 5′UTR-specific m6A and its involvement in cerebral cortical development and brain disorders from a novel perspective.

Download and Analysis of Raw Sequencing Data

The m6A-seq data of mouse embryonic (the 12.5–13-day embryo, E12.5–13) and postnatal (the 14-day offspring, P14) cerebral cortices were downloaded from the NCBI data bank (NCBI GEO: GSE141938). According to a previous study [25], the achieved data were pretreated and filtered. Briefly, the cleaned reads were achieved from the raw m6A-seq data by trimming the adapter using trim galore, followed by the alignment to the house mouse (Mus musculus) genome UCSC mm10. The PCR duplicates in the aligned data were removed by the MarkDuplicates function of Picard (version 1.95) with parameters, REMOVE_DUPLICATES = Ture. After filtering the duplicates, same tools were used to sort the bam file according to the coordinate. These m6A peaks were detected by MACS (version 1.4.0rc 2) with parameters, -format = “BAM” – gsize = 282,000,000 – tsize = 101 – nomodel – shiftsize = 150 – to-large False -w -S. The significant 6 mA peaks were then defined with the parameters of Peakscore ≥500, FoldEnrichment ≥2.0, and p value ≤0.001. Since each m6A peak was converged to about 100-nucleotides wide fragment with a summit in the center, we analyzed the distribution of the m6A-binding site by the localization of the summit. For example, if a center summit of m6A peak was localized within the 5′UTR of a transcribed RNA, this m6A-binding site was termed as 5′UTR specificity, and the RNA was defined as a 5′UTR-m6A-methylated RNA.

Moreover, the transcriptomic data of mouse embryonic (E12.5–13) and postnatal (P14) cerebral cortices were downloaded from the NCBI data bank (NCBI GEO: GSE116056). All raw reads were filtered and normalized as fragments per kilobase of exon per million fragments mapped (FPKM). Genes were annotated according to the data mapped to the genome of the house mouse (M. musculus). In addition, genes with the value of log2 ([postnatal FPKM]/[embryonic FPKM]) ≥1.0 and of p value ≤0.05 were defined as the differentially expressed genes.

Enrichment of m6A-Modified Gene in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes

To analyze the regulatory role of m6A during neurogenesis, the m6A-modified RNAs were clustered into different categories by the DAVID online tool (https://david.ncifcrf.gov/) [33]. Using the Gene Ontology (GO) knowledgebase, the functions of m6A-modified RNAs were stated and classified into three categories, including BP, CC, and molecular function (MF). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (https://www.kegg.jp/kegg/) was performed to seek the key signaling pathways that m6A-modified RNAs were involved in [34]. All the categories were selected at the significant value of p < 0.05. Moreover, the presence of these results was all performed by the OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/tools). A full list of all targeted terms of the BP, CCs, MFs, and KEGG pathway category is provided in online supplementary Table 2 (for all online suppl. material, see www.karger.com/doi/10.1159/000521620).

View of the m6A Methylation Profile in Each Targeted Gene

The integrative genomics viewer (IGV) tool was used for the visualization of m6A peaks along the whole transcript [35]. Notably, we identified the position where the mean IGV value of peak was more than 3 approximately equal to log10 (500) (500 was the threshold of peakscores) as a significant methylation site. Even though the IGV presentation showed a visible peak, the site was considered as nonmethylated, if the IGV value was less than 3.

5′UTR-Specific m6A Profiles in Embryonic and Postnatal Cerebral Cortices

As m6A modification marks the tempo of cerebral corticogenesis [6], we used the m6A-RIP-seq data from mouse E12.5–13 and P14 cerebral cortices (NCBI GEO: GSE141938) to investigate the dynamic profile of 5′UTR-specific m6A in the developing cortex. Based on the solid data that were demonstrated by the previous study [30], we identified 428 and 425 RNAs with 5′UTR-specific m6A from embryonic stage (E-stage) cortices and postnatal stage (P-stage) cortices, respectively (online suppl. Table S1). Among those, there are 220 RNAs temporal-specifically showing the 5′UTR-m6A methylation at the embryonic stage (shown in Fig. 1a), while 229 at the postnatal stage (shown in Fig. 1b). These RNAs were termed as 5′UTR-m6A-unique RNAs. The rest RNAs were found to be m6A-methylated at 5′UTR as well as at the coding sequence (CDS), 3′UTR, or NSC region. Moreover, the dynamic change of 5′UTR-unique m6A in each RNA was analyzed by the comparison between those RNAs at E-stage cortices and those at P-stage ones. As a result, 86 RNAs were 5′UTR-m6A-methylated at the embryonic stage but demethylated at the postnatal stage, which was termed as embryonic-specifically methylated RNAs (E-SMRs) (shown in Fig. 1c). On the contrary, 95 RNAs were methylated at the 5′UTR-unique m6A site at the postnatal stage only and termed as P-SMRs (shown in Fig. 1c). Hundred and thirty-four RNAs were 5′UTR-uniquely m6A-methylated throughout cortical development and termed as continuously methylated RNAs (CMRs) (shown in Fig. 1c). In the following description, E-SMRs, P-SMRs, and CMRs represented RNAs with 5′UTR-unique m6A methylation, unless there existed special notes. In addition, we also analyzed the distribution of 5′UTR-m6A-methylated RNAs in each chromosome. As shown in online supplementary Figure S1, the proportion of 5′UTR-m6A-methylated RNAs in each chromosome ranged from 2.13% to 11.35%, compared to total 5′UTR-m6A-methylated RNAs (shown in online suppl. Fig. S1). Except for genes in the Y chromosome, genes in the other 20 chromosomes could be m6A-methylated within the 5′UTR of their transcripts. Collectively, these data suggest that RNA 5′UTR-unique m6A are methylated in a common and dynamic model and might play an essential role in the developing cerebral cortex.

Fig. 1.

Profiles of 5′UTR-unique m6A in the developing cerebral cortices. a Numbers of RNAs that were uniquely m6A-methylated in their 5′UTR at the embryonic stage. b Numbers of RNAs that were uniquely m6A-methylated in their 5′UTR at the postnatal stage. c Accounts of E-SMRs, annotated as E-SMRs; postnatal-specifically methylated RNAs, termed as P-SMRs; and CMRs at both stages, named as CMRs. All these RNAs were uniquely m6A-methylated within their 5′UTRs. d Heatmap diagram revealed the change of transcription level of targeted E-SMRs, P-SMRs, and CMRs from the E-stage to the P-stage, respectively.

Fig. 1.

Profiles of 5′UTR-unique m6A in the developing cerebral cortices. a Numbers of RNAs that were uniquely m6A-methylated in their 5′UTR at the embryonic stage. b Numbers of RNAs that were uniquely m6A-methylated in their 5′UTR at the postnatal stage. c Accounts of E-SMRs, annotated as E-SMRs; postnatal-specifically methylated RNAs, termed as P-SMRs; and CMRs at both stages, named as CMRs. All these RNAs were uniquely m6A-methylated within their 5′UTRs. d Heatmap diagram revealed the change of transcription level of targeted E-SMRs, P-SMRs, and CMRs from the E-stage to the P-stage, respectively.

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Furthermore, the expression level of all RNAs with 5′UTR-unique m6A was identified according to the RNA sequencing data of E- and P-stage cortices (NCBI GEO: GSE116056). As shown in Figure 1d, E-SMRs were generally downregulated from the E-stage to P-stage (37% downregulated E-SMRs vs. 17% upregulated E-SMRs), whereas most of P-SMRs were increased in the P-stage (17% downregulated P-SMRs vs. 51% upregulated P-SMRs) (shown in Fig. 1d). In addition, we found that the amounts of CMRs with upregulated or downregulated expression were close to each other (23% downregulated CMRs vs. 20% upregulated CMRs) (shown in Fig. 1d). Our results suggest that the 5′UTR-m6A methylation is a common event throughout the developing cortex, but it also presents a transcription-related preference to the temporal-specific RNAs.

Specific RNA Functions Associated with 5′UTR-Unique m6A

To highlight the specific role of 5′UTR-unique m6A methylation, we first defined the function of related RNAs by the GO analysis (online suppl. Table S2). Seven BPs were enriched by E-SMRs and mainly involved in transcription and multicellular organism development (shown Fig. 2a; online suppl. Table S2). Among those enriched BPs, 5′UTR-unique E-SMRs shared common terms of DNA-templated transcription (GO: 0006351 and 0006355), cell differentiation (GO: 0030154), multicellular organism development (GO: 0007275), and transforming growth factor-beta receptor signaling pathway (GO: 0007179) with the outcomes of 3′UTR- or NSC-specific E-SMRs (online suppl. Table S2). On the contrary, two out of seven BPs were exclusively found and termed as mRNA transport (GO: 0051028) and mRNA processing (GO: 0006397) in the 5′UTR-m5A-associated enrichment (shown in Fig. 2a). Intriguingly, most of the E-SMRs in these two processes showed no significant change in their transcript level from the embryonic stage to the postnatal stage (shown in Fig. 2a; online suppl. Table S2). These data suggest that 5′UTR-m6A methylation, rather than the transcription level, plays a predominant role in regulating mRNA transport and processing. Meanwhile, seven out of nine BP terms were uniquely obtained by the enrichment of P-SMRs (shown in Fig. 2b; online suppl. Table S2). These terms were generally associated with the transmembrane process, such as neurotransmitter transport (GO: 0006836), gamma-aminobutyric acid transport (GO: 0015812), and positive regulation of dopamine secretion (GO: 0033603). Only two BP terms, chemical synaptic transmission (GO: 0007268) and dephosphorylation (GO: 0016311), were commonly enriched by both 5′UTR-unique P-SMRs and 3′UTR- or NSC-specific P-SMRs (online suppl. Table S2). In addition, the P-SMR-enriched BP terms generally consisted of more upregulated RNAs, except for the processes of detoxification of copper ion (GO: 0010273) and gamma-aminobutyric acid transport (GO: 0015812) (shown in Fig. 2b [the]; online suppl. Table S2). CMRs were enriched in fourteen unique BP terms, which were generally associated with protein-related processes (i.e., peptidyl-threonine phosphorylation, dephosphorylation, protein export from nucleus, Ras protein signal transduction, and protein localization to the plasma membrane) (shown in Fig. 2c; online suppl. Table S2).

Fig. 2.

Functional analysis of transcribed RNAs with 5′UTR-unique m6A methylation. a Dynamic enrichment circle diagram for E-SMRs. b, c Dynamic enrichment circle diagram for P-SMRs and CMRs, respectively. The annotation for each ring frame of circle diagrams was presented in online suppl. Figure S2.

Fig. 2.

Functional analysis of transcribed RNAs with 5′UTR-unique m6A methylation. a Dynamic enrichment circle diagram for E-SMRs. b, c Dynamic enrichment circle diagram for P-SMRs and CMRs, respectively. The annotation for each ring frame of circle diagrams was presented in online suppl. Figure S2.

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Moreover, analysis of unique CC revealed that E-SMRs preferred to act as the components of the subcellular unit, like ribonucleoprotein (GO: 1990904), focal adhesion (GO: 0005925), and Golgi apparatus (GO: 0005794) (shown in Fig. 2a; online suppl. Table S2). Meanwhile, the P-SMRs were differentially involved in the constitution of specific-type cell, including neuron projection (GO: 0043005), neuronal cell body (GO: 0043025), and postsynaptic density (GO: 0014069) (shown in Fig. 2b; online suppl. Table S2). Notably, we found that Olig2, an oligodendrocyte marker, was 5′UTR-uniquely methylated at the postnatal stage and involved in the cytoplasm (GO: 0005737) (online suppl. Table S2). Besides, Satb2, a marker for differentiated neuron in neocortices, was found to be postnatal-specific methylated at both 5′UTR and 3′UTR (online suppl. Table S1). Although no cell subtype-specific RNAs belonged to 5′UTR-unique E-SMRs in CC terms, Eomes (annotated as Tbr2, an intermediate progenitor marker) could be found to harbor m6A methylation sites at both 5′UTR and 3′UTR. Moreover, CMRs were more likely to be parts of extracellular components, including the extracellular exosome (GO: 0070062) and vesicle (GO: 1903561) (shown in Fig. 2c; online suppl. Table S2). These results indicate that 5′UTR-m6A methylation was more likely to modulate the development of common cells at embryonic stage, but it contributes more to the development of neural-specific cell at postnatal stage. In addition, the transcript level of each term was reflected in the purple circle of the diagram (shown in Fig. 2). Although half of E-SMRs in CC terms were differentially expressed from embryonic to postnatal stage, the other half showed no significant changes in their transcript level (shown in Fig. 2a). On contrary, most of P-SMRs in CC terms were upregulated. These data suggest that the transcript level was much more involved in the regulation of CCs at postnatal stage. We also performed the MF analysis and found that uniquely enriched terms for E-SMRs were associated with RNA binding (GO: 0003729, 0003723 and 00044822), and those for P-SMRs were involved in phosphatase activity (GO: 0004721, 0016791, and 0004865) (shown in Fig. 2a, b; online suppl. Table S2). Meanwhile, CMRs were uniquely enriched in three MF terms, including snRNA binding (GO: 0017069), GTPase activity (GO: 0003924), and calmodulin-dependent protein phosphatase activity (GO: 0033192) (shown in Fig. 2c; online suppl. Table S2). Overall, our results suggest that 5′UTR-specific m6A methylation is involved in distinct cell processes at different stages of the developing cortex and also performs a unique role in neurogenesis compared to those of 3′UTR-specific m6A methylation.

Signaling Pathways Specifically Enriched by RNAs with 5′UTR-Unique m6A

To further clarify the unique way that 5′UTR-unique m6A participates in, we examined the signaling pathways with 5′UTR-m6A-methylated RNAs during cortical development. Using the KEGG analysis, we enriched two pathways for E-SMRs, which were termed as RNA transport and mRNA surveillance pathway (shown in Fig. 3a; online suppl. Table S2). Both pathways were not achieved by 3′UTR-specific E-SMRs and thus appeared to be the unique outcomes of 5′UTR-unique E-SMRs enrichment. Surprisingly, none of significant pathways was enriched by P-SMRs (show in Fig. 3b; online suppl. Table S2). These results indicate a limited contribution of 5′UTR-unique m6A methylation to biological pathways during temporal-specific development of cortices. In addition, CMRs were enriched in 21 pathways, many of which were signaling pathways (online suppl. Table S2). Among those, nine pathways were specifically enriched by 5′UTR-unique CMRs (shown in Fig. 3c), of which the most significant pathways were dopaminergic synapse and chemokine signaling pathways. These data implicate that these two pathways were continuously modulated by 5′UTR-m6A methylation and might influence the cortical development throughout the embryonic and postnatal stages. Similarly, the 5′UTR-m6A methylation showed a persisting regulatory role in the other seven pathways, which might be involved in brain function disorders, such as prion disease, morphine addition, and Chagas disease.

Fig. 3.

Collection of the most impacted KEGG terms in the developing cortices. a 5′UTR-unique E-SMRs were enriched in two pathways, including RNA transport and mRNA surveillance pathways. Both pathways were uniquely enriched by 5′UTR-unique E-SMRs but not by 3′UTR- or NSC-specific E-SMRs. b 5′UTR-unique P-SMRs show no significant enrichment in KEGG analysis. c Nine out of twenty-two pathways were uniquely enriched by 5′UTR-unique CMRs and performed in the diagram.

Fig. 3.

Collection of the most impacted KEGG terms in the developing cortices. a 5′UTR-unique E-SMRs were enriched in two pathways, including RNA transport and mRNA surveillance pathways. Both pathways were uniquely enriched by 5′UTR-unique E-SMRs but not by 3′UTR- or NSC-specific E-SMRs. b 5′UTR-unique P-SMRs show no significant enrichment in KEGG analysis. c Nine out of twenty-two pathways were uniquely enriched by 5′UTR-unique CMRs and performed in the diagram.

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Status of 5′UTR-m5A-Methylation in E-SMRs Involved in the Enriched Pathways

The finely tuned RNA transport and surveillance are necessary for neurogenesis and neural repair, as well as neurological disorders [36‒38]. Taken together with the E-SMRs enrichment results, we are promoted to examine the 5′UTR-specific m6A status in genes among E-SMRs in these two pathways. As a result, we uncovered five genes that are involved in RNA transport and mRNA surveillance pathways (shown in Fig. 4; online suppl. Table S2). As shown by the IGV, Nup62, a component of the central channel of the nuclear pore complex, harbored one m6A-binding site within its 5′UTR (shown in Fig. 4). Alyref2 and Nxt1 are two members of the exon-junction complex and showed two and one m6A-binding sites, respectively. Cleavage stimulation factor (Cstf3) is a member of the Cstf3 complex and also harbored only one m6A-binding site. Similarly, Fmr1 showed only one m6A-binding site within its 5′UTR (shown in Fig. 4). We also found that only Nxt1 was downregulated from the embryonic stage to postnatal stage, and the rest showed no significant change in their transcription level (shown in Fig. 4). These data highlight a much greater role of 5′UTR-m6A modification in regulating genes in RNA transport and surveillance, while the transcription pattern contributes less to the processes.

Fig. 4.

Status of 5′UTR-unique m6A methylation sites of genes in the pathways of RNA transport and surveillance. 5′UTR-m6A-methylated patterns of five targeted E-SMRs (Nup62, Alyref2, Cstf3, Nxt1, and Fmr1) were showed by IGV analysis. Among those, Nup62is a member of the NPC. In addition, Alyref2is an outer shell of the EJC, while Nxt1acts as a transiently interacting factor in EJC. Cstf3is a component of the CstF complex. In this illumination, the red, green, and yellow boxes indicate the transcription level of a targeted gene as upregulation, downregulation, or no significant change from embryonic cortices to postnatal ones. NPC, nuclear pore complex; EJC, exon-junction complex.

Fig. 4.

Status of 5′UTR-unique m6A methylation sites of genes in the pathways of RNA transport and surveillance. 5′UTR-m6A-methylated patterns of five targeted E-SMRs (Nup62, Alyref2, Cstf3, Nxt1, and Fmr1) were showed by IGV analysis. Among those, Nup62is a member of the NPC. In addition, Alyref2is an outer shell of the EJC, while Nxt1acts as a transiently interacting factor in EJC. Cstf3is a component of the CstF complex. In this illumination, the red, green, and yellow boxes indicate the transcription level of a targeted gene as upregulation, downregulation, or no significant change from embryonic cortices to postnatal ones. NPC, nuclear pore complex; EJC, exon-junction complex.

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The 5′UTR-Specific m6A Patterns in RNAs Related to Neurological Disorders

A previous study analyzed the association of 3′UTR-m6A-methylated genes with neurological disorders [30]; hence, we examined whether 5′UTR-m6A-methylated genes were associated differently with neurological disorders. Notably, none of 5′UTR-m6A uniquely methylated RNAs was defined as a pathogenic gene. Thus, all 5′UTR-m6A-methylated RNAs were considered and used here, including those with an m6A-binding site in their CDS, 3′UTR, or NSC region as well. As compared to the reported pathogenic genes of neurological disorders [39], we identified only six disease risk genes modified by m6A methylation that is restricted to the 5′UTR (shown in Fig. 5; Table 1). Among those, Ep300, Ccnd2, Arx, and Eomes belonged to the 5′UTR-unique E-SMRs, and each gene harbored one m6A-binding site within the 5′UTR (shown in Fig. 5a). Two disease risk genes, Foxg1 and Mapt, were found to be 5′UTR-m6A-methylated CMRs, and both showed one 5′UTR-specific m6A-binding site (shown in Fig. 5b). Notably, none of P-SMRs was identified as known disease risk gene. In addition, these genes were associated with microcephaly (Ep300 and Foxg1), megalencephaly (Ccnd2), lissencephaly (Arx), polymicrogyria (Eomes), and Parkinson disease (Mapt), respectively (Table 1). To deeply understand the potential mechanism of 5′UTR-m6A-methylated genes in brain disorder, an online analysis tool (KOBAS-i) was used for the performance of gene set enrichment [40]. We found that Ep300 and Ccnd2 were two crucial genes and broadly participated in 25 and 26 pathways, respectively (Table 1). Among those, cell cycle, Wnt signaling pathway, FoxO signaling pathway, and Jak-STAT signaling pathway were enriched by both genes (online suppl. Table S3). In spite that Eomes shows no detectable signaling pathway among our analysis, Arx, Foxg1, and Mapt were found to participate in at least one or two pathways (Table 1, online suppl. Table S3). Interestingly, the FoxO signaling pathway was a common pathway enriched by Ep300, Ccnd2, and Foxg1, indicating that the FoxO signaling pathway might be an important way for 5′UTR-m6A-methylated genes to regulate neural development and function.

Table 1.

The temporal-specific methylation type of 5′UTR-m6A-methylated genes that are associated with neurological disorders and their involvement in KEGG pathways

 The temporal-specific methylation type of 5′UTR-m6A-methylated genes that are associated with neurological disorders and their involvement in KEGG pathways
 The temporal-specific methylation type of 5′UTR-m6A-methylated genes that are associated with neurological disorders and their involvement in KEGG pathways
Fig. 5.

Status of 5′UTR-m6A methylation sites of genes in neurological disorders. a IGV analysis of four 5′UTR-specific E-SMRs that act as pathogenetic genes (Ep300, Ccnd2, Arx, and Eomes) associated with neurological disorders. b IGV analysis of two 5′UTR-specific CMRs (Foxg1and Mapt).

Fig. 5.

Status of 5′UTR-m6A methylation sites of genes in neurological disorders. a IGV analysis of four 5′UTR-specific E-SMRs that act as pathogenetic genes (Ep300, Ccnd2, Arx, and Eomes) associated with neurological disorders. b IGV analysis of two 5′UTR-specific CMRs (Foxg1and Mapt).

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Because the FoxO signaling pathway plays a neuroprotective role [41, 42], we also examined 5′UTR-specific m6A status in genes in this pathway. Besides Ep300, Ccnd2, and Foxg1, there are other nine genes to be 5′UTR-m6A-methylated in the FoxO signaling pathway. As shown in Figure 6, each gene harbored only one m6A-binding site within their 5′UTR (shown in Fig. 6). We found that all these targeted genes belonged to either the E-SMRs cluster or CMRs cluster, except for Homer1 defined as a P-SMR (online suppl. Table S4). We also noticed that Stk11, Akt, Foxo4, Ccnd2, and Cdkn1b were downregulated, indicating that their transcription level was relatively higher at the embryonic stage. Meanwhile, Kras, Mapk1, Homer1, and Agap2 were downregulated, suggesting their lower transcription level in embryonic cortices (shown in Fig. 6). Interestingly, most of downregulated genes play positive roles in the activation of the FoxO signaling pathway, while the upregulated genes act as repressors to this signaling pathway. These results suggest that the FoxO signaling pathway was transcriptionally activated at embryonic cortices, which was cohesively regulated by 5′UTR-m6A methylation as well. In addition, Foxg1, a neurodevelopmental signal responsory, was continuously methylated within its 5′UTR and showed no significant change of the expression level from the E-stage to P-stage. Collectively, our results indicate that the FoxO signaling pathway is activated by differentially expressed genes at the embryonic stage, and 5′UTR-specific m6A methylation is a specific modification to modulate these genes at the posttranscriptional level.

Fig. 6.

Status of 5′UTR-specific m6A methylation sites of genes in the FoxO signaling pathway. Using IGV analysis, twelve 5′UTR-m6A-methylated genes were selected and performed. Among those, seven genes were defined as CMRs (Stk11, Akt, Foxo4, Agap2, Mapk1, Foxg1, and Grb2), while four genes belonged to E-SMRs (Ccnd2, Cdkn1b, Kras, and Ep300) and one gene was P-SMR (Homer1). In this illumination, the red, green, and yellow boxes indicate the transcription level of a targeted gene as upregulation, downregulation, or no significant change from embryonic cortices to postnatal ones.

Fig. 6.

Status of 5′UTR-specific m6A methylation sites of genes in the FoxO signaling pathway. Using IGV analysis, twelve 5′UTR-m6A-methylated genes were selected and performed. Among those, seven genes were defined as CMRs (Stk11, Akt, Foxo4, Agap2, Mapk1, Foxg1, and Grb2), while four genes belonged to E-SMRs (Ccnd2, Cdkn1b, Kras, and Ep300) and one gene was P-SMR (Homer1). In this illumination, the red, green, and yellow boxes indicate the transcription level of a targeted gene as upregulation, downregulation, or no significant change from embryonic cortices to postnatal ones.

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The proper development of the cerebral cortex is dependent on the fine-tuning control of gene expression, which is associated with both the transcription level and posttranscriptional modification [43, 44]. m6A plays a predominant role in posttranscriptional modification and mainly appears within the 5′UTR, CDS, or 3′UTR [27, 31, 45]. Recently, more studies have focused on the specific role of m6A at different RNA regions [28, 30, 31]. In this study, we specifically analyzed the unique contribution of 5′UTR-specific m6A methylation in mouse embryonic and postnatal cortices using MeRIP sequencing data. Excluding the similar functions shared by 3′UTR- or NSC-specific m6A, 5′UTR-unique m6A methylation plays a distinct but limited role in the temporal development of cerebral cortices and neurological disorders. Although further experimental evidence is needed, our study reveals the unique function of m6A in the 5′UTR and provides a fundamental reference for understanding 5′UTR-specific m6A methylation in the developing cortex.

In consistent with previous reports that m6A modification shows preference to 3′UTR and NSC region [27, 28], our analysis performed major distribution of m6A in the CDS region, 3′UTR, and/or NSC region. Only 8% of m6A-modified RNAs harbored m6A-binding sites within the 5′UTR. Among those, 27% and 30% of RNAs were temporal-specifically methylated at E-stage (E-SMRs) and P-stage (P-SMRs), respectively. These results implicate that 5′UTR-specific m6A methylation is a common but limited modification in the developing cortices. Previous studies have shown that 5′UTR-specific m6A methylation was responsible for translation in a 5′ cap-independent manner [31, 32]. In our comprehensive analysis, the transcription level of 5′UTR-specific E-SMRs, as well as P-SMRs, was partially higher in their corresponding stages, suggesting that 5′UTR-specific m6A modification might show more correlation with the translation level of RNAs than with their transcription level. Taken together, our study highlights the main function of 5′UTR-specific m6A, which is to influence RNA expression in the developing mouse cortices through posttranscriptional regulation.

Moreover, the unique function of 5′UTR-m6A-modified RNAs was analyzed as compared to those of 3′UTR-m6A-modified RNAs in the developing cortices. Similar to 3′UTR-m6A-modified RNAs [30], 5′UTR-m6A-modified RNAs function differently at the embryonic or postnatal stage. For instance, 5′UTR-unique E-SMRs mainly participated in cellular development and RNA processing, while 5′UTR-unique P-SMRs were involved in protein modification and signal transportation. On the other hand, there also exists a functional difference between 5′UTR-m6A-modified RNAs and 3′UTR-m6A-modified ones in both embryonic and postnatal cerebral cortices. Being different from 3′UTR- or NSC-specific E-SMRs [30], the 5′UTR-specific E-SMRs were uniquely enriched in mRNA processing and transport. Similarly, specific inhibition of m7G-cap methylation in the liver was demonstrated to delay the RNA processing by inhibiting cap-binding complex association [2]. These results illuminate an important role of 5′UTR-m6A methylation in mRNA 5′terminal-related events. Moreover, the enrichment of RNA transport was consistent with the result of later KEGG analysis. Recently, Ru W.X. and his collaborators have summarized a mechanism of m6A-regulating mRNA export, which depended on the recognition of m6A reader YTHDC1 [46]. In our study, we offered a novel potential mechanism for the 5′UTR-m6A regulation of mRNA transport, which is based on the alternative expression of related genes in the developing cortices. First, both GO and KEGG analysis targeted five 5′UTR-unique E-SMRs, which were responsible for mRNA recognition and export. Second, the transcriptomic analysis revealed that most of these E-SMRs were not significantly changed in their transcription level, suggesting that transcription pattern is not the important effector in regulating gene expression. Hence, 5′UTR-m6A methylation might play a predominant role in the regulation of gene expression, which further affected the efficiency of mRNA transport in embryonic cortices. In addition, studies have demonstrated that m6A methylation was usually associated with mRNA splicing or stability, when it occurred in the CDS or 3′UTR [3, 5]. Here, 5′UTR-specific m6A might also affect mRNA splicing or stability through modifying Alyref2 and Nxt1 (two proteins that interact with the mRNA-exon junction) and Cstf3 (a factor related to the cleavage of the polyadenylated tail). As compared within region-specific P-SMRs, 5′UTR-unique P-SMRs were uniquely enriched in protein dephosphorylation, which was not found in 3′UTR- or NSC-specific P-SMRs [30]. Considering the fact that decreasing the m6A level could enhance the p53 phosphorylation [47], we supposed that 5′UTR-m6A methylation might be more likely to contribute to the dephosphorylation of protein, for example p53, in postnatal cortices. Besides, 5′UTR-m6A-methylated RNAs showed a limited influence on substrate transport, while 3′UTR-m6A-methylated RNAs were widely associated with diverse signal transport [30]. The limited function of 5′UTR-unique m6A might be attributed to the adverse distribution preference in the 5′UTR of RNAs. In addition, the transcription level of RNAs involved in these BPs was also differentially changed. Surprisingly, we found that 5′UTR-specific P-SMRs showed no unique pathway enrichment, indicating that 5′UTR-specific m6A showed a more specific role in embryonic cortices rather than in postnatal ones. Taken together, these results suggest that 5′UTR-specific m6A methylation and RNA transcription patterns parallelly controlled the cortical developmental progress.

As a previous study has reported the association between m6A-methylated genes and neurological disorders, there raises the question of whether and how 5′UTR-m6A-methylated genes were involved in brain disorders. Because of the limited accounts of 5′UTR-m6A-methylated genes, we could not found any pathogenic gene that was m6A-methylated uniquely in the 5′UTR. Thus, we detected all 5′UTR-m6A-methylated genes and found six disease risk genes. Among those, Ep300 was demonstrated to regulate the proliferation and differentiation of neural stem cells during adult neurogenesis in zebra fish [48]. However, Ep300 was 5′UTR-m6A-methylated specifically at mouse embryonic cortices but not postnatal cortices. These results suggest that either 5′UTR-m6A methylation plays a repressive role in embryonic Ep300 function or there exists a functional difference of Ep300 between mouse and zebra fish. A similar situation happened to Arx, which is defined as a marker of adult neural stem cells [49] but was 5′UTR-m6A-methylated in embryonic cortices. However, Ccnd2 and Eomes (also annotated as Tbr2) are essential for the proliferation of the intermediate progenitor cell in the embryonic cortex [50, 51], which is inconsistent with their m6A-modified type of embryo-specific methylation within the 5′UTR. In addition, Ccnd2 showed a wide involvement in the diverse signaling pathways, such as the Wnt signaling pathway and Hippo signaling pathway. The aberrance of each pathway could cause dysregulation of migrating neurons and polarity of cortical neural progenitors [52, 53]. Similarly, 5′UTR-specific m6A also acts as a potential mediator of the expression of Foxg1 and Mapt, which were tightly related to cortical development and disorders [54, 55]. Moreover, half of the targeted disease risk genes were shown to be involved in the FoxO signaling pathway, suggesting its special role in the determination of neurological disorders. Besides, the FoxO signaling pathway was demonstrated to mainly act as a neuro-protector against cerebral ischemia [41], indicating that 5′UTR-m6A-methylated RNAs have a specific role in neuroprotection. Studies that 5′UTR-specific m6A methylation was responsible for diverse cellular stresses [31, 32] further supported our deduction. Moreover, our transcriptomic analysis revealed that the relatively higher transcription level of Stk11, Akt, Foxo4, Ccnd2, and Cdkn1b in embryonic cortices could facilitate the FoxO signaling pathway in cell cycle control, while the lower transcription level of Kras, Mapk1, Homer1, and Agap2 could relieve their repressive effect on the FoxO signaling pathway at the embryonic stage. Importantly, most of these genes were 5′UTR-m6A-methylated either at the embryonic-specific stage or throughout the developmental period of mouse cerebral cortices. These results suggest that m6A methylation patterns, coordinating with the transcription patterns, play important roles in the determination of neurological disease via the FoxO signaling pathway, in which, the m6A methylation pattern makes more contributions at the embryonic stage. Besides the FoxO signaling pathway, other pathways of CMRs-enriched KEGG were also closely related to neurological disorders. These phenomena implicate that the aberrance of 5′UTR-m6A methylation at any stages could lead to brain disorders through the dysregulation of dopaminergic synapse, chemokine signaling pathway, and/or other pathways. Our analysis highlights the possible way of 5′UTR-specific m6A to participate in neurological disorders. Even though we deduced the important and unique role of 5′UTR-specific m6A, solid experimental evidences were further required to support our conclusion.

Collectively, our study has specifically revealed the pattern of 5′UTR-specific m6A in the developing mouse cerebral cortex and uncovered its unique role in different neurological processes. Our finding has given a novel view of m6A methylation within the RNA 5′UTR in the developing cerebral cortex and provided an informative reference for future mechanistic studies of 5′UTR-specific m6A in normal brain development and neurological disorders.

No animal and no patient experiments were performed in this study. The Animal Care and Use Committee of College of Biological Science and Engineering, Fuzhou University (2019-SG-007), had made the decision that the ethics approval was not required in this case.

The authors declare that there is no conflict of interest associated with this study.

This study is funded by the National Natural Science Foundation of China (grant no. 81701132), the Natural Science Foundation of Fujian (grant no. 2018J05058), the Priming Scientific Research Foundation of Fuzhou University, China (grant. no. GXRC-20007, 510863), and the Foundation of Marine Bioenzyme Engineering Innovation Service Platform (grant no. 2014FJPT02).

Long-Bin Zhang and Ting-Ting Qiu conceived and designed the study. Long-Bin Zhang performed major analysis of the data. Ting-Ting Qiu and Wu-Wei-Jie Yang assisted the performance. Long-Bin Zhang, Ting-Ting Qiu, and Wu-Wei-Jie Yang interpreted the data and wrote the paper, and all the authors provided feedback. All the authors have read and agreed to the published version of the manuscript.

The data that support the findings of this study are openly available in the NCBI data bank at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141938 and at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi for the m6A-RIP-seq data of mouse embryonic (E12.5–13) and postnatal (P14) cerebral cortices (NCBI GEO: GSE141938) [30] and corresponding transcriptomic data (NCBI GEO: GSE116056) [56], respectively.

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