The Molecular and Function Characterization of Porcine MID2

SIMPLE SUMMARY: Midline2 (MID2) is widely involved in development, innate immunity, cancer, viral infections, and disease, but its role in pigs remains unexplored. In this study, we describe the structure and function of porcine MID2. Our results revealed that the gene of pMID2 is highly conserved with human and other vertebrate animals and its expression is started by NF-κB. pMID2 is distributed in cell membrane and cytoplasm and widely expressed in pig lung, spleen, and other organs. Moreover, pMID2 could enhance the activity of the ISRE promoter, and is mainly involved in the JAK-STAT signaling pathway. The above findings provide useful information for studying the function of pMID2 in pigs. ABSTRACT: Midline2 (MID2/TRIM1) is a member of the tripartite motif-containing (TRIM) family, which is involved in a wide range of cellular processes. However, fundamental studies on porcine MID2 (pMID2) are still lacking. In this study, we identified and characterized the full length MID2 gene of pig (Sus scrofa). The sequence alignment analysis results showed that pMID2 had an N-terminal RING zinc-finger domain, BBC domain, and C-terminal COS box, FN3 motif, and PRY-SPRY domain that were conserved and similar to those of other vertebrates. Furthermore, pMID2 had the highest expression levels in porcine lung and spleen. Serial deletion and site-directed mutagenesis showed that the putative nuclear factor-κB (NF-κB) binding site may be an essential transcription factor for regulating the transcription expression of pMID2. Furthermore, the immunofluorescence assay indicated that pMID2 presented in the cell membrane and cytoplasm. To further study the functions of pMID2, we identified and determined its potential ability to perceive poly (I:C) and IFN-α stimulation. Stimulation experiments showed pMID2 enhanced poly (I:C)-/IFN-α-induced JAK-STAT signaling pathway, indicating that pMID2 might participate in the immune responses. In conclusion, we systematically and comprehensively analyzed the characterizations and functions of pMID2, which provide valuable information to explore the pMID2 functions in innate immunity. Our findings not only enrich the current knowledge of MID2 in IFN signaling regulation but also offer the basis for future research of pig MID2 gene.