Cooperative roles of Pm-RORα splice variants in modulating the immune response in the pearl oyster P. f. martensii.

RAR-related orphan receptor alpha (RORα), a core member of the NR1F subgroup within the nuclear receptor superfamily, critically regulates lipid metabolism and immune responses. This study identified the Pm-RORα (Pm-RORα1 and Pm-RORα2) gene and its alternative splicing isoforms in Pinctada fucata martensii and investigated their functions through bioinformatic analysis, quantitative PCR (qPCR), and bimolecular fluorescence complementation (BiFC). Phylogenetic analysis revealed high homology between Pm-RORα and the Pacific oyster Crassostrea gigas ROR. Pm-RORα1_1 and Pm-RORα1_2 were generated by exon skipping of exons 2 and 6 in Pm-RORα1, respectively, while intron retention in exon 7 of Pm-RORα2 produced Pm-RORα2_1. Domain analysis showed that Pm-RORα1_1 and Pm-RORα1_2 lacked the ZnF_C4 and HOLI domains, respectively, while Pm-RORα2_1 was deficient in the HOLI domain. qPCR analysis demonstrated the expression patterns following LPS stimulation, Pm-RORα1_1 and Pm-RORα2_1 were significantly upregulated at 12 h, whereas Pm-RORα1_2 peaked at 6 h. Following nucleus transplantation, stimulation induced significant upregulation of Pm-RORα1, Pm-RORα1_1 and Pm-RORα1_2 at 6 h, while both of Pm-RORα2 and Pm-RORα2_1 was significantly downregulated from 6 to 24 h. Bimolecular fluorescence complementation assays confirmed protein interactions between Pm-RORα1 and the three isoforms (Pm-RORα1_1, Pm-RORα1_2, and Pm-RORα2_1). These findings indicate that Pm-RORα and its splicing isoforms potentially form protein interactions to modulate dynamic immune regulation in P. f. martensii, providing molecular insights into molluscan innate immunity and pearl cultivation optimization.