Cloning of the RNA m6A Methyltransferase 3 and Its Impact on the Proliferation and Differentiation of Quail Myoblasts

**Background:** N6-methyladenosine (m6A) modification is the most common RNA modification in eukaryotic cells and plays important roles in RNA processing, transport, translation, and stem cell differentiation. Methyltransferase 3 (METTL3) is the key enzyme catalyzing m6A modification. While METTL3's role in mammalian myoblast proliferation and differentiation has been studied, its function in poultry skeletal muscle development was unknown. The quail industry has broad development prospects due to quails' small size, short breeding cycle, and high nutritional value, making understanding the genetic mechanisms of poultry skeletal muscle development economically important.

**Methods:** The authors obtained 220 fertilized Korean Longcheng quail eggs and incubated them at 38°C with 60% relative humidity. Leg muscle tissue was collected daily from embryonic day 7 (E7) through post-hatch day 1 (P1). Sex was determined by PCR amplification of the CHD1 gene (63 female, 55 male embryos identified). The full-length CDS of quail METTL3 was obtained via 3' rapid amplification of cDNA ends (RACE) using gene-specific primers based on NCBI reference sequence XM_015850477.2. The assembled CDS was synthesized and inserted into pEGFP-C1-3×flag vector. QM7 cells were cultured in M199 medium with 10% FBS, 10% tryptose phosphate broth, and 0.5% penicillin/streptomycin at 37°C with 5% CO2. Differentiation was induced with M199 medium containing 2% horse serum. Cell proliferation was assessed by CCK-8 assay (absorbance at 450 nm) and cell cycle analysis by flow cytometry. Gene expression was measured by qPCR using the 2^−∆∆Ct method with GAPDH as internal control. RNA-seq was performed on an Illumina Novaseq 6000 System (PE150), and data were analyzed with GSEA (significance: |NES| > 1, NOM.pval < 0.05, FDR.qval < 0.05).

**Key Results:** The 3' RACE yielded an 889-bp sequence, and splicing produced a METTL3 mRNA with a CDS of 1683 bp encoding a 560 amino acid protein. Western blotting confirmed the quail METTL3 protein size at approximately 70 kDa (100 kDa with 3×flag-eGFP fusion). During quail leg muscle development, MYOG, MYOD, and MSTN expression peaked at E9-E10, and METTL3 expression was relatively high at E7-E8, peaked at E9, and gradually decreased thereafter. In QM7 cells, METTL3 overexpression (approximately 150-fold increase, p < 0.001) significantly decreased the proportion of cells in G1 phase (p < 0.001) and increased those in S phase (p < 0.01). METTL3 knockdown significantly decreased the proportion of cells in G2 phase (p < 0.01). CCK-8 assays showed METTL3 overexpression significantly increased proliferation (p < 0.001), while knockdown decreased it (p < 0.01). METTL3 overexpression significantly increased MYOG, MYOD1, and MEF2C expression, while knockdown produced opposite effects. RNA-seq identified 138 differentially expressed genes (80 upregulated, 58 downregulated) after METTL3 overexpression. KEGG enrichment showed significant enrichment in 14 pathways including MAPK signaling pathway-fly, vascular smooth muscle contraction, and lipid metabolism. GSEA identified significant changes in MANNOSE_TYPE_O_GLYCAN_BIOSYNTHESIS, RIBOSOME, and PENTOSE_PHOSPHATE_PATHWAY pathways.

**Clinical Implications:** This study provides the first full-length CDS of quail METTL3 and demonstrates its pro-proliferative and pro-differentiation effects on quail myoblasts. The findings establish METTL3-mediated RNA m6A modification as an important epigenetic regulatory mechanism in poultry skeletal muscle development. Understanding this regulatory network could inform breeding strategies to improve meat production traits in the poultry industry. The study also highlights the multidimensional regulatory role of METTL3, which controls multiple genes and signaling pathways (including MAPK and ribosome pathways) in myoblast biology.