Detection of global RNA modifications and analysis of m6A modifications in sheep hearts at different altitudes

Research on ruminant adaptation to high-altitude hypoxia has increasingly expanded into the field of epigenetic regulation. Among various RNA modifications, N 6 -methyladenosine (m 6 A) plays an important role in the hypoxic adaptation of the heart. However, systematic characterization of RNA modification profiles and m 6 A regulatory networks in sheep from different altitude-associated groups remains limited. Therefore, this study aimed to investigate cardiac RNA modification patterns and the expression of key m 6 A regulatory factors in sheep from different altitude-associated groups. This study profiled RNA modifications and m 6 A-related regulatory factors in left ventricular myocardial tissues from low-altitude Hu sheep, mid-altitude Tibetan sheep, and high-altitude Tibetan sheep. Among 55 targeted RNA modifications, 25 modified nucleosides were detected in sheep heart RNA. Levels of 2’-O-methyladenosine (Am) and m 6 A were significantly higher in Hu-low than in Tib-mid. At the protein level, Wilms tumor 1-associated protein (WTAP), fat mass and obesity-associated protein (FTO), YTH domain family protein 2 (YTHDF2), and YTH domain family protein 3 (YTHDF3) were significantly upregulated in Tib-mid. YTH domain family protein 1 (YTHDF1), YTH domain containing protein 1 (YTHDC1), and YTH domain containing protein 2 (YTHDC2) were more highly expressed in Hu-low and Tib-mid than in Tib-high, whereas no significant differences were observed for methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), or AlkB homologue 5 (ALKBH5). At the transcriptional level, WTAP , METTL3 , FTO , and all examined reader genes were significantly upregulated in Tib-mid. Because altitude and breed background were confounded, comparisons involving Hu-low should be interpreted cautiously. This study provides a descriptive profile of cardiac RNA modifications and m 6 A regulator expression in sheep from different altitude-associated backgrounds. The observed differences may reflect combined effects of altitude-related environment and breed-specific genetic background rather than altitude alone. These findings suggest that m 6 A-related pathways may be associated with cardiac adaptation to hypoxic environments and provide candidate targets for future functional validation.