Roles Of Shrimp MiR-1in Phagocytosis Regulation And Characterization Of Shrimp SiRNA Binding Protein

MicroRNAs are a class of endogenous non-coding RNAs with20-24nt, which has been widely confirmed to be involved in most biological processes, including cell development, cell differentiation, metabolism and tumorigenesis. To characterize the miRNAs related to phagocytosis in Marsupenaeus japonicus shrimp, the cytochalasin B was injected into shrimp in this study. By miRNAs deep sequencing analysis, a total of21miRNAs involved in phagocytosis regulation of shrimp were obtained. Among the miRNAs involved in phagocytosis, miR-1was a conserved miRNA from invertebrates to mammals. The results showed that the miR-1could regulate phagocytosis in shrimp and RAW264.7macrophage. Our data indicated that the miR-1played a crucial role during phagocytosis by targeting clathrin heavy chain. Our study would be very helpful to reveal the regulatory mechanism mediated by miRNA in phagocytosis.RNAi is a gene-silencing process triggered by double-stranded RNAs (dsRNAs), which can cause the cleavage of homologous mRNA. In RNAi, the short interfering RNAs (siRNAs),-21-25nt double-stranded fragments, serve as the core mediators of RNAi and are produced from dsRNAs by Dicer. The siRNAs accompanied with the argonaute family proteins (Ago proteins) and some auxiliary proteins comprise the RNA-induced silencing complex (RISC) which directly mediates target gene degradation. The process of RISC assembly includes sophisticated steps, in which many proteins are involved except for argonaute proteins. However, the proteins interacted with siRNA are not extensively explored. Our previous study shows that the vp28-siRNA is a21nt short interfering RNA that targets envelope protein vp28gene of white spot syndrome virus (WSSV). In this study, the vp28-siRNA was characterized to reveal the proteins bound with it. Based on the biotin/streptavidin affinity screening, it was found that the shrimp arginine kinase was specifically bound with the vp28-siRNA. The co-immunoprecipitation assays revealed that the siRNA was interacted with arginine kinase, suggesting that arginine kinase was an essential component of RNA-induced silencing complex. Therefore our study presented a novel finding on the RISC components, which would be helpful to reveal the molecular events in the RNAi pathway.