Comprehensive profiling analysis of the N6-methyladenosine-modified circular RNA transcriptome in cultured cells infected with Marek's disease virus

Abstract

Marek's disease virus (MDV) induces severe immunosuppression and lymphomagenesis in the chicken, its natural host, and results in a condition that investigated the pathogenesis of MDV and have begun to focus on the expression profiling of circular RNAs (circRNAs). However, little is known about how the expression of circRNAs is referred to as Marek's disease. Previous reports have is regulated during MDV replication. Here, we carried out a comprehensive profiling analysis of N6-methyladenosine (m⁶A) modification on the circRNA transcriptome in infected and uninfected chicken embryonic fibroblast (CEF) cells. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) revealed that m⁶A modification was highly conserved in circRNAs. Comparing to the uninfected group, the number of peaks and conserved motifs were not significantly different in cells that were infected with MDV, although reduced abundance of circRNA m⁶A modifications. However, gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses revealed that the insulin signaling pathway was associated with the regulation of m⁶A modified circRNAs in MDV infection. This is the first report to describe alterations in the transcriptome-wide profiling of m⁶A modified circRNAs in MDV-infected CEF cells.

Figure 1

Flowchart depicting construction of cDNA libraries used for m⁶A-modified circRNA transcriptome sequencing of uninfected and Md5-infected CEF cells. (Figure was made by Adobe Illustrator, v 24.0, https://www.adobe.com/cn/products/illustrator.html).

Figure 2

(A) Differentially expressed circRNAs in infected and control groups. The X axis represents the expression level of the gene in the control group, and the Y axis represents the expression level of the gene in the infected group. A represents the uninfected group and B represents the infected group. Red dots represent up-regulated genes in group B compared with group A. Green dots represent down-regulated genes in group B compared with group A. Blue dots represent genes with no significant differences in group B compared with group A. (Made by edgeR, v3.16.5, http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) (B,C) GO enrichment of host genes associated with down-regulated circRNAs for (B) biological processes and (C) molecular functions; (D) KEGG pathway analysis of the host genes for circRNAs.

Figure 3

The characteristics of m⁶A peaks. (A) Venn diagram of m⁶A genes in infected Md5 and CEF groups; (B) Venn diagram of m⁶A methylation sites identified in circRNAs from infected and CEF groups; (C) The sequence motif of m6A sites in infected and control groups (Made by Discriminative Regular Expression Motif Elicitation (DREME), v5.3.3, https://meme-suite.org/meme/tools/dreme); (D) Proportion of genes harboring different numbers of m⁶A sites in the two groups (Made by Prism, v8.0.2, https://www.graphpad.com).

Figure 4

m⁶A modification clustering analysis. (A) Cluster analysis of m⁶A methylation at the transcriptome level. (B) Cluster analysis of m⁶A m6A modified lncRNA genes in the infected and control groups. The color represents the size of the log-fold enrichment (FE) value: the closer the color is to red, the larger the logFE value. Md5-1, Md5-2, Md5-3 represent the Md5-infected CEF group with three independent replicates. ( Made by Tbtools, v1.082, http://www.tbtools.org).

Figure 5

Chromosome visualization of m⁶A sites in lncRNAs. Red represents the infected group while blue represents the control group. Md5-1, Md5-2, Md5-3 represent the Md5-infected CEF group with three independent replicates. Differentially methylated m⁶A peaks visualized in Md5-infected group and control group. The highlighted area represents one of the differential methylation peaks.

Figure 6

Gene ontology analyses of the infected and control groups. (A) The top ten gene ontology terms of biological processes were significantly enriched for the up-regulated genes; (B) The top seven gene ontology terms of cell component were significantly enriched for the up-regulated genes; (C) The top ten gene ontology terms of molecular functions were significantly enriched for the up-regulated genes; (D) The top ten gene ontology terms of biological process significantly enriched for down-regulated genes; (E) The top five gene ontology terms of molecular functions significantly enriched for down-regulated genes.

Figure 7

KEGG pathway analysis of differentially methylated m⁶A genes in circRNAs. (A) Bar plot showing the top 10 enrichment scores of significantly enriched pathways for up-methylated m⁶A genes in the infected group; (B) Bar plot showing the top 10 enrichment scores of significantly enriched pathways for down-methylated m⁶A genes in the infected group.