The Dicer from oyster Crassostrea gigas functions as an intracellular recognition molecule and effector in anti-viral immunity

Abstract

Dicer, as a member of ribonuclease III family, functions in RNA interference (RNAi) pathway to direct sequence-specific degradation of cognate mRNA. It plays important roles in antiviral immunity and production of microRNAs. In the present study, a Dicer gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDicer) of 1873 amino acids containing two conserved ribonuclease III domains (RIBOc) and a double-stranded RNA-binding motif (DSRM). The deduced amino acid sequence of CgDicer shared identities ranging from 18.5% to 46.6% with that of other identified Dicers. The mRNA transcripts of CgDicer were detectable in all the examined tissues of adult oysters, with the highest expression in hemocytes (11.21 ± 1.64 fold of that in mantle, p < 0.05). The mRNA expression level of CgDicer in hemocytes was significantly up-regulated (36.70 ± 11.10 fold, p < 0.01) after the oysters were treated with double-stranded RNA (dsRNA). In the primarily cultured oyster hemocytes, the mRNA transcripts of CgDicer were significantly induced at 12 h after the stimulation with poly(I:C), which were 2.04-fold (p < 0.05) higher than that in control group. Immunocytochemistry assay revealed that CgDicer proteins were mainly distributed in the cytoplasm of hemocytes. The two most important functional domains of CgDicer, DSRM and RIBOc, were recombinant expressed in Escherichia coli transetta (DE3), and the recombinant DSRM protein displayed significantly binding activity to dsRNA and poly(I:C) in vitro, while the recombinant RIBOc protein exhibited significantly dsRNase activity to cleave dsRNA in vitro. These results collectively suggested that CgDicer functioned as either an intracellular recognition molecule to bind dsRNA or an effector with ribonuclease activity, which might play a crucial role in anti-viral immunity of oyster.