Fig 2: Knockout of DUSP11 decreases the accumulation of BLV 5p miRNAs. (A) Northern blot analysis of BLV-B2 and BLV-B5 miRNAs in parental (wild-type [WT]), DUSP11 knockout (D11-KO), and NoDice-2-20 (Dicer-KO) HEK293T cells. Cells were cotransfected with transfection control vector, pHSUR4, and each BLV miRNA expression vector. The membrane was first blotted with probes specific for the 5p miRNAs, stripped, reprobed for the 3p miRNAs, stripped, and reprobed for HSUR4 RNA. (B) Next-generation small RNA sequencing in parental (wild-type) and DUSP11 knockout HEK293T cells transiently expressing the BLV-B2 and BLV-B5 miRNAs. Bars represent the average fold change (DUSP11 knockout/wild-type) in a number of small RNA reads of the most abundant isoform of each indicated miRNA (Supplemental Fig. S3A) from two independent experiments ±the standard deviation. n = 2. The fold change of the 5p miRNAs was normalized to the fold change of the corresponding 3p miRNAs. (C) Western blot analysis of DUSP11 in parental FLK-BLV cells (fetal lamb kidney cells persistently infected with BLV) and various FLK-BLV-DUSP11 knockout cell lines (D11KO-1, D11KO-20, and D11KO-37). (D) Northern analysis of the BLV-B5 miRNAs in the parental, D11KO-1, D11-KO-20, and D11KO-37 FLK-BLV cell lines. The membrane was first probed for the 3p miRNA, stripped, and reprobed for the 5p miRNA. (E) Quantitation of the relative band density (5p/3p) of the BLV-B5 miRNAs by Northern blot analysis. Bars represent the average ± the standard deviation from three experiments. Values were normalized to the parental cells. The P-values were calculated using one sample t-test. (*) P < 0.05; (**) P < 0.005.

Fig 3: Knockout of DUSP11 decreases the RISC activity of the BLV 5p miRNAs. (A) Schematic diagram of the BLV miRNA biogenesis pathway. (B) Diagram of the BLV-B5 miRNA RISC reporters. Two sites complementary to either the 5p miRNA (green boxes) or the 3p miRNA (orange boxes) were inserted into the 3′ untranslated region (UTR) of Renilla luciferase. In the B5-5p docking site mutant (DSM), point mutations were made in the sequence complementary to the B5-5p seed to interfere with RISC-mediated silencing. (C) Immunoblot analysis to confirm CRISPR/Cas9-mediated knockout of DUSP11 in DUSP11 knockout clone 16 (DUSP11-KO-16) HEK293T cells as compared with the parental (wild-type [WT]) HEK293T cells. (D) Luciferase assay to measure the RISC activity of BLV-B5 miRNAs in parental (wild-type) and DUSP11 knockout clone 16 HEK293T cells. Bars represent the mean luciferase ratio (Renilla/firefly) ± SEM normalized to pISK-EV and the vector 3′ UTR reporter. Transfections were performed in triplicate in each experiment. Eight experiments were performed for vector 3′ UTR, B5-5p RR, and B5-3p RR cotransfected with pISK-EV and pBLV-B5. Four experiments were performed for vector 3′ UTR, B5-5p RR, and B5-3p RR cotransfected with pBLV-B1. Three experiments were performed for the B5-5p DSM with all expression vectors. (E) Diagram of the Rluc reporter containing the majority of the ovine CECR1 3′ UTR (green box), which has five potential target sites of BLV(913)-miR-B5-5p. Point mutations in all potential B5-5p target sites were made (red ×) to generate the Rluc CECR1 3′ UTR control vector (CECR1 DSM). (F) Luciferase assay measuring the repression of the CECR1 3′ UTR and B5-5p DSM by BLV-B5-5p in wild-type and DUSP11 knockout HEK293T cells. Bars represent the mean luciferase ratio (Renilla/firefly) ± SEM from four experiments. Values were normalized to pISK-EV and the vector 3′ UTR reporter.

Fig 4: Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. (A) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. (B) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A. The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. (C) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for AGO4) and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. (D) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. (E) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.

Fig 5: DUSP11 promotes accumulation, AGO association, and RISC activity of AdV5 mivaRNAI-5p. (A) Northern blot analysis of the 5p and 3p miRNAs derived from AdV5 VA RNAI from parental (wild-type [WT]), DUSP11 knockout (D11-KO), DUSP11-KO-pLenti-D11, and DUSP11-KO-pLenti-DUSP11-CM HEK293T cells transfected with pVAI. The membrane was first probed for the 5p miRNA, stripped, and reprobed for the 3p miRNA. (B) AGO RIP analysis of VAI-derived miRNAs. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pcDNA-EV, pAGO1-Flag, or pAGO2-Flag and either cotransfected with pVAI or infected with AdV5. RIP was performed 48 h after transfection using anti-Flag M2 antibody. One percent of the input RNA and 95% of RIP-recovered RNA were subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA, stripped, and reprobed for the 3p miRNA. Immunoblot analysis was performed on 0.1% of input lysate and 5% of the Flag RIP. (C) Luciferase assay to measure RISC activity of the 5p miRNA derived from VAI RNA in parental (wild-type), DUSP11 knockout, DUSP11-KO-pLenti-D11, and DUSP11-KO-pLenti-DUSP11-CM HEK293T cells. Bars represent the mean luciferase ratio (Renilla/firefly) ± SEM from three experiments (wild-type and DUSP11 knockout) or four experiments (stable cell lines) in which transfections were performed in triplicates.