Epitranscriptomic Analysis of m6A Methylome After Peripheral Nerve Injury

Abstract

N6-methyladenosine (m6A) is one of the most plentiful internal RNA modifications, especially in eukaryotic messenger RNA (mRNA), which plays pivotal roles in the regulation of mRNA life cycle and nerve development. However, the mRNA m6A methylation pattern in peripheral nervous injury (PNI) has not been investigated. In this study, sciatic nerve samples were collected from 7 days after sciatic nerve injury (SNI) and control rats. Quantitative real-time PCR demonstrated that m6A-related methyltransferase/demethylase genes were remarkably upregulated in SNI group compared with control group. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was performed to reveal the m6A methylation landscape. The results showed that 4,014 m6A peaks were significantly altered, including 2,144 upregulated and 1,870 downregulated m6A peaks, which were corresponded to 1,858 genes. Moreover, 919 differentially expressed genes were identified by the conjoint analysis of MeRIP-seq and RNA-seq. GO and KEGG pathway analyses were performed to determine the biological functions and signaling pathways of the m6A-modified genes. Notably, these genes were mainly related to the immune system process, cell activation, and nervous system development in GO analysis. KEGG pathway analysis revealed that these genes were involved in the cell cycle, B cell receptor signaling pathway, axon guidance pathway, and calcium signaling pathway. Furthermore, the m6A methylation and protein expression levels of autophagy-related gene (Atg7) were increased, together with the activation of autophagy. These findings shed some light on the epigenetic regulation of gene expression, which may provide a new opinion to promote functional recovery after PNI.

Keywords: RNA methylation; autophagy; axon regeneration; m6A; peripheral nerve.

FIGURE 1

The expression levels of writers and erasers. Quantitative RT-PCR showing the upregulated levels of METTL3, METTL14, WTAP, FTO, and ALKBH5. Among them, the expression levels of METTL14 and FTO were the most significant. GAPDH was used as an internal control. SNI group (n = 3); control group (n = 3). “**” and “***” indicate p < 0.01 and p < 0.001, respectively, via Student’s t test.

FIGURE 2

The degree and pattern of m6A modification peaks in SNI and control groups. (A) Volcano plots indicating the distribution of differential m6A peaks after SNI. Fold-change ≥ 2.0, p < 0.00001, n = 3, via Fisher’s exact test. (B) Priority region and average distribution of m6A peaks. (C,D) The distribution of m6A peaks in SNI and control groups, respectively. (E) The top motifs enriched across differential m6A peaks. (F) Data visualization of the m6A-modified Gtse1 mRNA.

FIGURE 3

GO and KEGG pathway analyses of the differentially methylated genes. Significantly enriched GO terms of the hypermethylated (A) and hypomethylated genes (B). Significantly enriched KEGG pathways of the hypermethylated (C) and hypomethylated (D) genes.

FIGURE 4

Conjoint analysis of m6A methylation and mRNA expression after SNI. (A) Volcano plots and (B) Heatmap plots displaying the differentially expressed genes between SNI and control groups. (C) Four-quadrant graph showing the association between mRNA m6A methylation and transcriptome level.

FIGURE 5

The m6A methylation and protein expression of Atg7 gene are altered, and autophagy is activated following PNI. (A,B) Integrative genomics viewer and gene-specific m6A qPCR demonstrating the increased methylation of Atg7 gene after SNI. n = 3, via Student’s t test. (C) Upregulation of ATG7 protein level. n = 3, via Western blot analysis. (D) Expression level of the autophagy-related marker LC3 is increased. n = 3. (E) Quantification of Western blots for ATG7 and LC3II/LC3I. n = 3, via ANOVA and post hoc analysis. (F) Transmission electron microscopy showing autophagosome (yellow arrow) and lysosome (blue arrow) in the sciatic nerve tissues. “*,” “**,” and “***” indicate p < 0.05, p < 0.01, and p < 0.001, respectively.