Role of NUDT3 in skeletal myogenesis and association of regulatory genetic variants with carcass traits in pigs

Skeletal muscle is central to locomotor function and metabolic regulation in humans and constitutes a primary source of high-quality protein in livestock production. While genome-wide association studies (GWAS) have identified a strong association between Nudix-type hydrolase 3 (NUDT3) and lean mass in both human (Homo sapiens) and pig (Sus scrofa) populations, its functional role in myogenesis remains unclear. In the present study, silencing of NUDT3 in porcine muscle satellite cells (PMSCs) markedly impaired proliferative capacity, as evidenced by G₁ stage accumulation and reduction in EdU⁺ cells. Concomitantly, knockdown of NUDT3 significantly suppressed myogenic differentiation, while NUDT3 overexpression yielded the opposite effects. In vivo, overexpression of NUDT3 in tibialis anterior (TA) muscle of mice (Mus musculus) significantly increased the abundance of Pax7⁺ and eMyHC⁺ cells following cardiotoxin-induced injury, thereby promoting regenerative myogenesis. Transcriptomic profiling demonstrated that NUDT3 significantly attenuated NF-κB signaling in a Nudix motif-dependent manner, providing mechanistic insight into its pro-myogenic function. Integrative co-localization of GWAS and expression quantitative trait locus (eQTL) signals identified eight single nucleotide polymorphisms (SNPs) significantly associated with NUDT3 expression in porcine muscle, including four variants located within evolutionarily conserved E6 and E7 enhancer elements supported by HiCuT profiling of H3K27ac and CRISPR interference assays. Among these, rs327612517 within E7 showed significant association with loin muscle area and depth in PigBiobank data and modulated E7 enhancer activity, as demonstrated by dual-luciferase reporter and electrophoretic mobility shift assays. Collectively, these findings establish NUDT3 as a positive regulator of skeletal myogenesis and identify rs327612517 as a functional variant with potential utility for genetic improvement of lean yield in pigs.