Fig 1: Analysis of cellular RNAs in DUSP11 knockout cell lines. (A) Host gene expression (RefSeq genes ≤500 nt with no annotated coding sequence) in HEK293T cells with or without Terminator treatment assayed by unfragmented TGIRT-seq. Read counts from the indicated cell line library mapping to annotated host genes in reads per million mapped (RPMM) are plotted on each axis. snoRNAs are indicated with blue circles. (B) Expression analysis of select host RNAP III transcribed genes in HEK293T, HEK293T DUSP11 knockout, A549, and A549 DUSP11 knockout cell lines assayed by fragmented TGIRT-seq with and without Terminator treatment. The log base 2 ratio of gene counts from the indicated libraries is plotted on each axis. (C) Northern blot analysis of candidate ncRNA DUSP11 targets from the indicated cell lines. EtBr-stained low-molecular-weight RNA is provided as an additional loading control. The membrane was first probed for vtRNA1-2, stripped, and reprobed for the indicated RNAs. (D) Model for the role of DUSP11 in RNA silencing and modulation of RNAP III transcripts in mammalian cells. RNAP III transcribed RNAs initially contain a 5′ triphosphate. DUSP11 dephosphorylates a fraction of these RNAs. This reduces the steady-state level and alters the activity/function of some RNAs. For RNAP III transcribed miRNA precursors, the 5p arm of the resulting 5′ monophosphorylated miRNA precursors is predominantly loaded into AGO proteins to generate stable/functional 5p RISCs. The miRNA precursors that remain 5′ triphosphorylated predominantly load the 3p miRNA in AGO, while the 5′ triphosphorylated 5p miRNAs are rapidly degraded. Furthermore, the 5′ triphosphorylated 5p miRNAs that are incorporated into AGO to generate an unstable 5p RISC, promoting degradation of the complex/5p miRNA. The red dashed arrow indicates the DUSP11-dependent, Dicer-independent route for the BLV 5p miRNAs and other noncanonical interfering RNAs.
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: DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. (A) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. (B) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A. Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. (C) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.
Fig 4: 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.
Fig 5: 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.
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