The role of N6-methyladenosine RNA methylation in the heat stress response of sheep (Ovis aries).

With the intensive development of the sheep industry and increasing global temperatures, heat stress in sheep has become an increasingly severe and important issue in recent years. The level of N6-methyladenosine (m6A) RNA methylation changes in response to stress plays important roles in stress responses. However, the role of m6A in the heat stress response of sheep remains unclear. To explore this issue, we measured heat stress protein (HSP) expression, liver function indexes, m6A on RNA, m6A-related enzyme expression, and tissue damage in sheep that had been subjected to heat stress. At the transcriptome level, our results showed significant increases in m6A on RNA and increased mRNA levels of HSPs (HSP70, HSP90, and HSP110) and m6A-related enzymes [METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), WTAP (wilms tumor 1-associated protein), FTO (fat mass and obesity-associated protein), ALKBH5 (alkB homologue 5), YTHDF1-3 (YTH domain family proteins), and YTHDC1-2 (YTH domain-containing proteins)] following heat stress. At the protein level, the expression of METTL3, YTHDF1-2, and YTHDC2 showed no significant differences following heat stress. However, in contrast to its mRNA level after heat stress, the protein expression of YTHDF3 was reduced, while the expression of HSPs (HSP70, HSP90, and HSP110), METTL14, WTAP, FTO, ALKBH5, YTHDF3, and YTHDC1 increased in line with their measured mRNA levels. Histological experiments revealed that heat stress caused varying degrees of damage to sheep liver tissue. Moreover, immunohistochemical staining indicated that the m6A-related enzymes were expressed in sheep hepatocytes, and differences in expression patterns were observed between the control and heat stress groups. In summary, differences in the level of m6A and the expression of m6A-related enzymes in the liver of sheep were observed after heat stress. This indicates that m6A is involved in the regulation of heat stress in sheep. Our findings provide a new avenue for studying the responses to heat stress in sheep.

Identification and Characterization of Long Noncoding RNAs in Ovine Skeletal Muscle.

Long noncoding RNAs (lncRNAs) are increasingly being recognized as key regulators in many cellular processes. However, few reports of them in livestock have been published. Here, we describe the identification and characterization of lncRNAs in ovine skeletal muscle. Eight libraries were constructed from the gastrocnemius muscle of fetal (days 85 and 120), newborn and adult Texel and Ujumqin sheep. The 2002 identified transcripts shared some characteristics, such as their number of exons, length and distribution. We also identified some coding genes near these lncRNA transcripts, which are particularly associated with transcriptional regulation- and development-related processes, suggesting that the lncRNAs are associated with muscle development. In addition, in pairwise comparisons between the libraries of the same stage in different breeds, a total of 967 transcripts were differentially expressed but just 15 differentially expressed lncRNAs were common to all stages. Among them, we found that TCONS_00013201 exhibited higher expression in Ujumqin samples, while TCONS_00006187 and TCONS_00083104 were higher in Texel samples. Moreover, TCONS_00044801, TCONS_00008482 and TCONS_00102859 were almost completely absent from Ujumqin samples. Our results suggest that differences in the expression of these lncRNAs may be associated with the muscular differences observed between Texel and Ujumqin sheep breeds.

MiR-378 and BMP-Smad can influence the proliferation of sheep myoblast.

MicroRNA (miRNA) is a sort of endogenous ~20-25 nt non-coding RNAs, and it can regulate a variety of biological events. We found the miR-378 may involve in regulating the muscle development of sheep during our previous research. However, the molecular mechanism of miR-378 regulating myoblast proliferation is still unclear. In this research, we predicted that BMP2 (Bone morphogenetic protein 2) was the target gene of miR-378 and the BMP-Smad signal pathway that BMP2 participated in playing an important role in the muscle development. Therefore, we tried to determine whether miR-378 influence myoblast proliferation of sheep through the BMP-Smad signal pathway. The results indicated that inhibit BMP-Smad signal pathway by interfering Smad4 to promote proliferation of sheep myoblasts; promote BMP-Smad signal pathway by interfering Smad7 to inhibit proliferation of sheep myoblasts; over-expression miR-378 promotes BMP-Smad signal pathway and myoblast proliferation in sheep; interfering miR-378 inhibits BMP-Smad signal pathway and myoblast proliferation in sheep. However, when both of which functioned at the myoblast, miR-378 could not fully depend on BMP-Smad signal pathway to regulate myoblast proliferation. In sum, both miR-378 and BMP-Smad can influence the proliferation of myoblast, but miR-378 does not target the 3' UTR of sheep BMP2.