Functional characterization of white spot syndrome virus-responsive miR-750 in black tiger shrimp Penaeus monodon

Abstract

In the RNA interference (RNAi) process, small RNAs called microRNAs (miRNAs) modulate gene expression post-transcriptionally by acting as guides to direct Argonaute (Ago)-containing RNA-induced silencing complexes (RISCs) to targeted mRNAs. The Penaeus monodon miR-750 was previously reported as the highly upregulated miRNA during white spot syndrome virus (WSSV) infection. In this study, the computational program and 2D gel electrophoresis technique were used to identify miR-750 target genes. Bioinformatic analysis suggested that the predicted target mRNAs of miR-750 were ubiquitin-conjugating enzyme E2 Q1 gene (UbcQ1), Actin1 (Act1) and Sarcoplasmic calcium-binding protein (Scp). Furthermore, the alteration of protein expression in the stomach as a result of introducing the miR-750 was investigated by 2-DE technique. The 5 downregulated protein spots upon enhancing of miR-750 mimic were identified as 4 types of protein including actin1 (Act1), hemocyanin (Hc), sarcoplasmic calcium-binding protein (Scp) and tropomyosin (Tpm) whereas the 8 upregulated protein spots were identified as 3 types of proteins such as actin1 (Act1), chymotrypsin (Ctr) and hemocyanin (Hc). The gene expression level of only Scp determined in WSSV-infected P. monodon stomach by qRT-PCR showed negative correlation to miR-750 expression. The dual luciferase reporter assay for miR-750 and Scp target sequences showed the reduced luciferase activities about 20%, respectively. From the above results, we could identify the miR-750 target as Scp. It is known that miRNA is loaded into the Ago family of proteins during miRNA biogenesis and then the miRNA-RISC complex (miRISC) binds complementarily to the target. Therefore, Ago contributes to the robustness of miRNA-mediated gene regulation. So far, there is no report about Ago function associated with miRNA biogenesis. We showed that PmAgo3 was involved in miR-750 biogenesis. The effect of introducing miR-750 mimic and miR-750 anti-miRNA oligonucleotide (AMO) into shrimp on the WSSV copy number, Scp gene expression and the expression of shrimp immune-related genes in stomach of WSSV-infected P. monodon was investigated. Overexpression of miR-750 led to low level of Scp gene expression and the significantly high level of WSSV copy number in WSSV-infected shrimp. During WSSV infection, overexpression of miR-750 led to the decrease in Pen5, PmCasp, and PmCaspase gene expression when compared with that of the control WSSV-infected group. Taken together, we can conclude that during WSSV infection, the upregulated miR-750 targets Scp led to alteration in expression of antimicrobial peptide and apoptosis genes and promoting viral infection.