Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei

Abstract

Dicer (Dcr) is the key protein of the RNA interference (RNAi) pathway. To investigate the role of the RNAi pathway in shrimp anti-viral immunity, Litopenaeus vannamei Dcr2 (designated as LvDcr2) was identified and characterized. The full-length cDNA of LvDcr2 was 5513 bp long, with an open reading frame encoding a putative protein of 1502 amino acids. In addition, five proteins homologous to the single von Willebrand factor type C (VWC) domain protein (SVC) were also identified in L. vannamei and named LvSVC1-5. These LvSVCs were between 102 and 190 amino acids in length and all contained a motif similar to Drosophila melanogaster SVC proteins (DmSVCs). By co-immunoprecipitation assays and pull-down assays, we demonstrated that LvDcr2, L. vannamei Argonaute 2 (LvAgo2), and L. vannamei transactivating response RNA-binding protein isoform 1 (LvTRBP1) interacted with each other. A luciferase reporter assay indicated that the promoters of LvSVC1, LvSVC4, LvSVC5, and DmSVC Vago ( DmVago) were activated by LvDcr2 as well as by Drosophila Dcr2 (DmDcr2). Real-time RT-PCR showed that LvDcr2 and LvSVCs were up-regulated in immune responses against Poly(C-G) or WSSV challenge. These results suggested that LvDcr2 formed complexes with LvAgo2 and LvTRBP1 to act as the cores of shrimp small interfering RNA (siRNA)-induced silencing complex (siRISC)/siRISC-loading complex (siRLC), role in shrimp siRNA pathway. Furthermore, these results also suggested that LvDcr2 may engage in non-specific activation of anti-viral immunity.