RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

Abstract

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.

Keywords: RNA editing; adenosine deaminase acting on RNA; miRNA-induced silencing complex; virus latency.

Figure 1.

Host and virus miRNAs and mRNAs in the Ago1 complex. (a) The copy number of WSSV genomic DNA in the haemocytes of virus-infected shrimp pre-infection (0), and 24 and 48 h post-infection was quantified using quantitative real-time PCR. (b) The Ago1 complex of virus-infected shrimp haemocytes was co-immunoprecipitated with Ago1-specific antibody. Then the complex was analysed by western blot analysis (i). The anti-GST antibody was used as a control. RNAs in the Ago1 complex were extracted (ii) and subjected to sequencing. M, protein marker or RNA marker. (c) Size distribution of small RNAs in the Ago1 complex pre-infection (0), and 24 and 48 h post-infection. (d) Percentages of sequence reads of shrimp and viral miRNAs in the Ago1 complex of shrimp pre-infection (0), and 24 and 48 h post-infection. (e) The shrimp miRNA (i) and WSSV-miRNA (ii) expression profiles of WSSV-challenged shrimp pre-infection (0), and 24 and 48 h post-infection The numbers on the right indicated the log₁₀ of the number of copies of miRNAs. (f) Northern blots of selected shrimp and WSSV miRNAs. Total RNAs extracted from the Ago1 complexes of virus-free or WSSV-infected shrimp haemocytes were blotted with the DIG-labelled oligodeoxynucleotide probes pre-infection (0), and 24 and 48 h post-infection. (g) Heat map of differentially expressed shrimp and WSSV genes in the Ago1 complex of shrimp haemocytes. The shrimp were infected with WSSV. mRNAs in the Ago1 complex of shrimp haemocytes were sequenced pre-infection (0), and 24 and 48 h post-infection. The colours indicate the Z-values of genes. (h) Real-time PCR detection of gene expression profiles of shrimp in response to WSSV pre-infection (0), and 24 and 48 h post-infection.

Figure 2.

Interactions between miRNAs and mRNAs in Ago1 complex of shrimp. (a) Distribution of target RNA-annotated clusters across transcripts from Ago1 complex of virus-free and virus-infected shrimp pre-infection (0), and 24 and 48 h post-infection. The numbers indicated the time points of virus infection. (b) Numbers of genes targeted by host and virus miRNAs detected at 0, 24 and 48 h post-infection. (c) Function and pathway analyses of genes targeted by miRNAs. GO was performed by comparing the coding sequences of the transcripts in the Ago1 complex with the GO database with the blast E-value of less than 1 × 10⁻⁵. KEGG classifications of the genes were simultaneously characterized. (d) Heat map generated from GO analysis of transcripts targeted by the top 20 miRNAs with high expression level. Tree showed the hierarchical clustering of miRNAs based on GO analysis. The colours indicated the significant differences between clusters.

Figure 3.

Viral miRNA editing mediated by host adenosine deaminase acting on RNA (ADAR). (a) Comparison of WSSV-miR-N12 mature sequence with the WSSV genomic DNA sequence. The editing site was coloured. (b) The predicted hairpin structure of WSSV-miRNA-N12 precursor using mFold software (http://frontend.bioinfo.rpi.edu/applications/mfold/). Red indicates the mature sequence of WSSV-miRNA-N12. Green indicates the potential A-to-I editing site. (c) Neighbour-joining phylogenetic tree analysis of ADAR proteins of vertebrates and invertebrates. Bootstrap values are shown. The bar represents the distance. hsa, Homo sapiens; mmu, Mus musculus; xtr, Xenopus (Silurana) tropicalis; gga, Gallus gallus; dre, Danio rerio; dme, Drosophila melanogaster; ame, Apis mellifera; cel, Caenorhabditis elegans; zne, Zootermopsis nevadensis. (d) Western blot analysis of the expressed shrimp ADAR protein in insect cells. The plasmid expressing the V5-ADAR fusion protein and the synthetic WSSV-miRNA-N12 precursor were cotransfected into insect cells. The synthetic WSSV-miRNA-N12 precursor alone was included in the transfection as a control. The expression of shrimp ADAR was detected by the V5 antibody. M, protein marker. (e) The involvement of shrimp ADAR in the RNA editing of viral miRNA. The total RNAs were extracted from insect cells transfected with the plasmid expressing the shrimp ADAR protein and/or the synthetic WSSV-miRNA-N12 precursor. Then the precursor of WSSV-miRNA-N12 was cloned and sequenced. The positions of the edited site and the unedited site are indicated with arrows.

Figure 4.

(Overleaf.) The role of viral miRNA editing in virus latency. (a) Viral miRNA detected in shrimp infected with WSSV. The expressions of edited and unedited WSSV-miR-N12 were examined by northern blot pre-infection (0 h), and post-infection (6, 12, 18, 24, 36 and 48 h). The percentages of edited and unedited viral miRNA were evaluated. (b) The silencing of viral miRNA expression. The expression of WSSV-miR-N12 in WSSV-infected shrimp was silenced by AMO-WSSV-miR-N12. The silencing was examined by northern blot pre-infection (0), and 12, 24, 36 and 48 h post-infection. U6 was used as a control. (c) Effects of WSSV-miR-N12 on the WSSV replication in shrimp. Shrimp were simultaneously injected with WSSV and AMO-WSSV-miR-N12. WSSV alone and AMO-WSSV-miR-N12-scrambled were used as controls. The shrimp were subjected to real-time PCR to detect the WSSV copies 12, 24, 36 and 48 h post-infection. The numbers indicated the time points post-infection. (d) Cumulative mortalities of WSSV-challenged shrimp after the injection of AMO-WSSV-miR-N12. Each point represented the mean of triplicate assays. (e) The overexpression of viral miRNA. The edited and unedited WSSV-miR-N12 mimics were co-injected with WSSV into shrimp, respectively. Edited and unedited WSSV-miR-N12 were detected pre-infection (0), and 12, 24, 36 and 48 h post-infection by northern blots. U6 was used as a control. (f) The shrimp were simultaneously injected with WSSV and the unedited or edited WSSV-miR-N12, and the WSSV copies in shrimp were monitored by quantitative real-time PCR at 12, 24, 36 and 48 h post-infection (g) The accumulative mortalities of WSSV-infected shrimp treated with the unedited or edited WSSV-miR-N12 were monitored 1–5 days post-infection. All the assays were repeated three times (*p < 0.05; **p < 0.01).

Figure 5.

(Overleaf.) The mechanism of viral miRNA editing in virus infection. (a) The region of the viral gene wsv399 3′UTR targeted by WSSV-miR-N12. The seed sequence of WSSV-miR-N12 is underlined. (b) Constructs of EGFP-wsv399-3′UTR and EGFP-wsv399-3′UTR-mutation. The sequence targeted by WSSV-miR-N12 is underlined. (c) Direct interaction between WSSV-miR-N12 and wsv399 gene in insect cells. Insect High Five cells were cotransfected with the WSSV-miR-N12 mimic or WSSV-miR-N12-mimic-scrambled and EGFP, EGFP-wsv399-3′UTR or EGFP-wsv399-3′UTR-mutation. At 36 h after cotransfection, the fluorescence of cells was examined. (d) The interaction between the edited WSSV-miR-N12 and wsv399 gene in insect cells. Insect High Five cells were cotransfected with the edited WSSV-miR-N12 mimic and EGFP-wsv399-3′UTR. WSSV-miR-N12-mimic-scrambled, EGFP and EGFP-wsv399-3′UTR-mutation were used as controls. At 36 h after cotransfection, the fluorescence of cells was evaluated. (e) The interaction between edited or unedited viral miRNA and host Ago1 protein. The unedited or edited WSSV-miRNA-N12 was incubated with recombinant shrimp Ago1 protein, then separated by native polyacrylamide gel and stained with ethidium bromide to visualize the miRNA (top), followed by staining with Coomassie blue (bottom). The wedges indicated the concentration gradient of recombinant protein used. (f) The interaction between viral miRNA and its target gene in the miRISC. The unedited or edited WSSV-miRNA-N12 and the 3′UTR of the wsv399 gene were incubated with shrimp Ago1 protein. Subsequently, the mixture was separated by agarose gel and stained with ethidium bromide to show the miRNA and target gene, followed by staining with Coomassie blue (bottom). The wedges indicate concentrations of recombinant protein used. (g) Northern blot analysis of expression profiles of the wsv399 gene in WSSV-infected shrimp. The shrimp were challenged with WSSV. Numbers indicated the time points post-infection. Shrimp β-actin was used as a control. (h) Silencing of wsv399 expression in WSSV-infected shrimp. The wsv399-siRNA and WSSV was co-injected into WSSV-infected shrimp. Then the shrimp haemolymph was subjected to northern blot analysis to detect the expression level of the wsv399 gene. Numbers indicate the time points post-infection. Shrimp β-actin was used as a control. (i) Effect of wsv399 gene silencing on WSSV copies in shrimp. The shrimp were simultaneously injected with WSSV and wsv399-siRNA, followed by detections of virus copies with quantitative real-time PCR. As controls, WSSV alone and wsv-399-siRNA-scrambled were included in the injections. (j) Accumulative mortalities of WSSV-infected shrimp. At different times post-infection, the accumulative mortalities of shrimp were monitored daily. Numbers showed the time points post-infection. (k) The detection of ADAR expression in virus-challenged shrimp. Shrimp were infected with WSSV. At different times post-infection, the ADAR mRNA level was evaluated with quantitative real-time PCR. (l) The examination of wsv399 expression. The wsv399 mRNA in shrimp treated with WSSV and unedited or edited WSSV-miR-N12 was quantified with quantitative real-time PCR. In all panels, the statistically significant differences between treatments were represented with asterisks (*p < 0.05; **p < 0.01).

Figure 6.

The model for the role of ADAR-mediated RNA editing of viral miRNA in virus–host interaction.