PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis

Abstract

MicroRNAs (miRNAs) are evolutionary conserved, non-coding small RNAs that have been shown to regulate diverse biological processes including immunity. In a previous study, a novel miRNA of Japanese flounder (Paralichthys olivaceus), pol-miR-novel_395, was found to be responsive in expression to the infection of the bacterial pathogen Edwardsiella tarda. In the present study, we examined the regulation and immune effect of pol-miR-novel_395 and its target gene. We found that pol-miR-novel_395 expression was regulated by E. tarda and megalocytivirus, and pol-miR-novel_395 targeted the gene of PUF60 (poly (U)-binding-splicing factor 60 kDa) of flounder (named PoPUF60). Constitutive expression of PoPUF60 occurred in relatively high levels in the heart and liver of flounder. Bacterial infection upregulated PoPUF60 expression, whereas viral infection downregulated PoPUF60 expression. Interference with PoPUF60 expression or overexpression of pol-miR-novel_395 in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoPUF60 enhanced bacterial dissemination in the tissues of flounder but blocked viral replication, whereas in vivo overexpression of PoPUF60 inhibited bacterial dissemination but facilitated viral replication. Additionally, pol-miR-novel_395 and PoPUF60 were involved in the process of autophagy and apoptosis. Collectively, these results indicated that PoPUF60 and pol-miR-novel_395 play an important role in pathogen infection, autophagy, and apoptosis.