The transcriptome-wide N6-methyladenosine (m6A) map profiling reveals the regulatory role of m6A in the yak ovary

Abstract

Background and aim: Yak estrus is a seasonal phenomenon, probably involving epigenetic regulation of synthesis and secretion of sex hormones as well as growth and development of follicles. N6-methyladenosine (m⁶A) is the most common internal modification of the eukaryotic mRNA. However, there are no detailed reports on the m⁶A transcriptome map of yak ovary. Therefore, this study aimed to collected the yak ovarian tissues at three different states of anestrus (YO-A), estrus (YO-F), and pregnancy (YO-P), and obtained the full transcriptome m⁶A map in yak by MeRIP-seq.

Results: The HE staining revealed that the number of growing follicles and mature follicles in the ovary during the estrus period was relatively higher than those in the anestrus period and the pregnancy period. The RT-qPCR showed that the expression of METTL3, METTL14, FTO, YTHDC1 were significantly different across different periods in the ovaries, which suggests that m⁶A may play a regulatory role in ovarian activity. Next, we identified 20,174, 19,747 and 13,523 m⁶A peaks in the three ovarian samples of YO-A, YO-F and YO-P using the methylated RNA immunoprecipitation sequencing (MeRIP-seq). The m⁶A peaks are highly enriched in the coding sequence (CDS) region and 3'untranslated region (3'UTR) as well as the conserved sequence of "RRACH." The GO, KEGG and GSEA analysis revealed the involvement of m⁶A in many physiological activities of the yak's ovary during reproductive cycle. The association analysis found that some genes such as BNC1, HOMER1, BMP15, BMP6, GPX3, and WNT11 were related to ovarian functions.

Conclusions: The comparison of the distribution patterns of methylation peaks in the ovarian tissues across different periods further explored the m⁶A markers related to the regulation of ovarian ovulation and follicular development in the yak ovary. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m⁶A modification in the yak ovary.

Keywords: Anestrus; Estrus; MeRIP-seq; Pregnancy; m6A.

Fig. 1

The microscopic observation of the tissue sections of the yak ovaries. YO-A, YO-F, and YO-P separately represent the yak ovary in the anestrus period, follicular (estrus) period, and pregnancy period

Fig. 2

The results of m⁶A methylase expression and genome sequencing. a The gene expression levels of 13 common m⁶A methylases on the three groups of YO-A, YO-F and YO-P. Different lowercase letters between two groups represent significant differences (p < 0.05), and the same letters among groups represent no significant differences (p > 0.05). b The principal component analysis of the three groups of samples. c The distribution of different types of reads in each sample was counted. d The valid data were matched to the reference genome defined as matched to exon, intron and intergenic. e The length distribution of m⁶A peaks in the YO-A, YO-F, and YO-P groups. f The relative expression levels of IP in the three groups of ovarian samples. g The distribution of m⁶A peak sites in the yak genome was obtained from three different samples. The outermost circle represents the chromosome distribution on the genome, the red circle represents the YO-A group, the purple circle represents the YO-F group, and the blue circle represents the YO-P group. h Density distribution of the m⁶A peaks in YO-A, YO-F and YO-P transcripts in the different gene structures

Fig. 3

Overview of the differential methylation peaks in the YO-FvsA, YO-FvsP, and YO-PvsA comparison groups. a The number of hypermethylation and hypomethylation in the YO-FvsA group. b The number of hypermethylation and hypomethylation in the YO-FvsP group. c The number of hypermethylation and hypomethylation in the YO-PvsA group. d The motif sequences “RRACH” were identified in the YO-FvsA, YO-FvsP, and YO-PvsA groups. e The four-quadrant diagram of the differentially expressed genes and m⁶A methylation genes in the YO-FvsA group. f The four-quadrant diagram of the differentially expressed genes and m⁶A methylation genes in the YO-FvsP group. g The four-quadrant diagram of differentially expressed genes and m⁶A methylation genes in the YO-PvsA group

Fig. 4

The gene qPCR results and transcriptome expression levels of BNC1, HOMER1, MMP16, P4HA3, BMP15, BMP6, ISG15, CCRL2, GPX3, WNT11, CYP1B1, and ITGA2 in the YO-A, YO-F and YO-P groups

Fig. 5

The enrichment plot of the GSEA results and the heat map of gene expression in each sample. a On the left is the enrichment plot of homologous recombination, on the right is the heat map of gene expression in YO-F and YO-A. b On the left is the enrichment plot of nuclear receptor activity, on the right is the heat map of gene expression in YO-F and YO-P. c On the left is the enrichment plot of gap junction, on the right is the heat map of gene expression in YO-P and YO-A