The novel fish miRNA pol-miR-novel_171 and its target gene FAM49B play a critical role in apoptosis and bacterial infection

Abstract

MicroRNAs (miRNAs) are a type of small, non-coding RNAs that participate in many cellular and biological processes by regulating mRNA stability. In a previous study, we identified 96 Japanese flounder (Paralichthys olivaceus) miRNAs responsive to the infection of Edwardsiella tarda, a bacterial pathogen to fish as well as humans. In the current study, we examined the regulation and function of one novel miRNA, i.e., pol-miR-novel_171, from the above 96 miRNA pool. We found that pol-miR-novel_171 expression was regulated by E. tarda and megalocytivirus in a pathogen-specific manner, and that pol-miR-novel_171 targeted the gene of FAM49B (family with sequence similarity 49 member B) of flounder (named PoFAM49B) by negative interaction with the 3′-UTR of PoFAM49B. To date, the function fish FAM49B is unknown. We found that PoFAM49B expressed in multiple tissues of flounder, and recombinant PoFAM49B interacted with and inhibited the growth of Gram-negative bacterial pathogens. Interference with PoFAM49B expression in flounder cells promoted E. tarda infection. Similar effects on E. tarda infection were observed with pol-miR-novel_171 overexpression. Consistently, in vivo knockdown of PoFAM49B in flounder enhanced E. tarda dissemination in fish tissues. Furthermore, interference with PoFAM49B expression, or overexpression of pol-miR-novel_171, promoted apoptosis of flounder cells, while in vitro and in vivo knockdown of PoFAM49B augmented the expressions of key apoptosis-associated genes. These results revealed for the first time the immune function of fish FAM49B and the regulatory mechanism of a novel fish miRNA by demonstrating that pol-miR-novel_171, via PoFAM49B, played a critical role in apoptosis and anti-bacterial immunity.