Integrative Analysis of MeRIP-Seq and RNA-Seq Reveals METTL14 as a Core Regulator of Follicular Granulosa Cell Development in Zi Geese

m6A is a ubiquitous reversible post-transcriptional RNA methylation modification in eukaryotic cells, which has been positive effect on regulating follicles development in animals. However, the role of m6A modification profiling in regulating the development of healthy and atresia small yellow follicle have not yet been studied in poultry. In this study, we conducted a comparative analysis of the m6A methylation profiles of healthy and atresia follicles Zi goose during the period of peak egg-laying. Here, we discovered that 23,342 and 25,552 m6A peak between healthy small yellow follicles group (HSYF) and atresia small yellow follicle groups (ASYF), which were mainly enriched in 3'-UTR and stop codon regions. We found that 1174 differential upregulated peaks and 1250 differential downregulated peaks were identified in ASYF group, these differential peaks were covered 1141 and 1233 genes, including METTL14, WTAP, IGF2BP3 and CYTB. Motif analysis demonstrated that these m6A peaks exhibit the RRACH and DRACH conserved consensus sequence. Importantly, Zi goose follice transcriptome was extensively methylated and a positive correlation between the m6A peak and gene expression levels. The combined analysis of MeRIP-seq and RNA-seq revealed that a total of 78 DMGs were shared in HSYF and ASYF groups, such as BMP5, PPARGC1A, NGF, SCD5, which were mainly involved in TGFβ signaling pathway, MAPK signaling pathway, PPAR signaling pathway and ECM receptor interaction. Furthermore, METTL14 plays a regulatory role in Zi goose granulosa cell development, which was verified by in vitro experiments. We found that knockdown of METTL14 dramatically prevented GCs apoptosis, promoted GCs proliferation, increased the production and secretion of steriod hormone, enhanced the expression levels of genes related to steroid hormone synthesis in granulosa cell. Conversely, overexpression of METTL14 resulted in opposite outcomes. Additionally, we also observed that knockdown of METTL14 increased the activities of antioxidant enzyme (SOD, GSH and CAT), decreased the activities of MDA in goose GCs. Conversely, overexpression of METTL14 inhibited the activities of antioxidant enzymes, increased the activities of MDA. In summary, these data collectively demonstrated that m6A methylation was widely distributed in the process of geese follicle growth and development, and futher confirm the significant role of METTL14 influences on granulosa cell development of Zi geese. These findings can be a considerable efficient way to faciliate the laying egg performance of Zi goose through molecular marker assisted breeding technology.