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. 2018 Dec 18;9:2999. doi: 10.3389/fimmu.2018.02999

Figure 5.

Figure 5

Underlying mechanism of the miR-1000-mediated simultaneous targeting of wsv191 and wsv407 mRNAs. (A) MiR-1000-mediated time-course degradation of wsv191 and wsv407 mRNAs. The 3′UTR of wsv191 mRNA or wsv407 mRNA and miR-1000 were incubated with shrimp Ago1 complex for different time intervals. The level of mRNA degradation was examined using Northern blot with wsv191-specific or wsv407-specific probes (up) and using agarose gel electrophoresis (down). Numbers indicated the incubation time. (B) Specificity of miR-1000-mediated degradation of target mRNAs. The 3′UTR of non-target gene wsv024 mRNA and miR-1000, as well as the wsv191 or wsv407 mRNA 3′UTR and miR-1000-scrambled, were incubated with shrimp Ago1 complex for different time intervals, followed by Northern blot with wsv024-specific probe (up), wsv191-specific probe or wsv407-specific probe (down). Numbers showed the incubation time. (C) MiR-1000-concentration dependent degradation of wsv191 mRNA or wsv407 mRNA. The 3′UTR of wsv191 mRNA or wsv407 mRNA and shrimp Ago1 complex were incubated with miR-1000 at different concentrations for 2 h. Then the mixture was subjected to Northern blot. (D) Viral mRNA stability analysis during WSSV infection. Shrimp were injected with WSSV. Forty eight h later, the WSSV-infected shrimp were treated with actinomycin D. At different time points after treatment, shrimp were subjected to Northern blotting to detect wsv191, wsv407, and wsv024 mRNAs. (E) MiR-1000-mediated synchronous degradation of wsv191 mRNA and wsv407mRNA. The 3′UTRs of wsv191 mRNA and wsv407 mRNA as well as miR-1000 were simultaneously incubated with shrimp Ago1 complex for different time intervals. Subsequently, the mixture was analyzed by Northern blot. (F,G) Competitive degradation assays of wsv191 and wsv407 mRNA 3′UTRs mediated by miR-1000. To assess the competitive degradation between wsv191 and wsv407 mRNA 3′UTRs, 10-fold concentration of wsv191 mRNA 3′UTR and 1-fold concentration of wsv407 mRNA 3′UTR (F) or 1-fold concentration of wsv191 mRNA 3′UTR and 10-fold concentration of wsv407 mRNA 3′UTR (G) were mixed and then incubated with miR-1000 together with shrimp Ago1 complex for different time intervals. The mixture was analyzed by Northern blot. (H) Sequencing of degraded 3′UTRs of wsv191 mRNA and wsv407 mRNA. The arrows indicated the sequences of wsv191 mRNA 3′UTR and wsv407 mRNA 3′UTR complementary to the seed sequence of miR-1000.