Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei.
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 5513bp 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.
MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, PR China.
Yi-Hong
YH
Chen
Xiao-Ting
XT
Jia
Li
L
Zhao
Chao-Zheng
CZ
Li
Shuang
S
Zhang
Yong-Gui
YG
Chen
Shao-Ping
SP
Weng
Jian-Guo
JG
He
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001685:Biological Factors / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D017930:Genes, Reporter; D047468:Immunoprecipitation; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D033561:Penaeidae / Q000201:enzymology* / Q000276:immunology / Q000821:virology; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation; D046848:White spot syndrome virus 1; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism; D000099253:Trans-Activation Responsive RNA-Binding Protein
D001685:Biological Factors; D049410:Luciferases, Renilla; D021382:Protein Sorting Signals; D016601:RNA-Binding Proteins; D043244:Ribonuclease III; D014841:von Willebrand Factor; D000099253:Trans-Activation Responsive RNA-Binding Protein