Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges

Abstract

Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.

Keywords: TuD; WPRE; decoy; miRNA sponge; miRNA suppression.

FIGURE 1.

Fusion of WPRE to TuDs facilitates enhanced miRNA suppression after transient plasmid transfections. (A) Schematic representation of TuDs expressed from the CMV RNA Pol II promoter and fused to eGFP and either the 3′-end (WPRE-TuD) or 5′-end (TuD-WPRE) of WPRE, eGFP and the 3′-end of antisense WPRE (WPRErev-TuD), or directly to eGFP (TuD). A schematic representation of the TuD hairpin is provided in Hollensen et al. (2013). (B) Levels of miR-7, -16, -21, and -203 suppression were measured by dual-luciferase assays using a reporter plasmid encoding an RLuc reporter gene made responsive to miRNA binding by a perfect miRNA target site. (C) Dual-luciferase assay comparing miRNA suppression by WPRE-TuD and TuD-WPRE. Data are depicted as mean + SEM. Asterisks above columns show P-values for comparison with the negative control WPRE-MCS cotransfected with psiCHECK-vector containing a perfect miRNA target site. Asterisks indicate significance as follows: (**) P < 0.01, (***) P < 0.001, (ns) not significant.

FIGURE 2.

WPRE in lentiviral vectors increases titers and miRNA suppression activity of TuDs. (A) Schematic representation of the lentiviral vector plasmid encoding eGFP- and WPRE-fused TuDs expressed from the PGK RNA Pol II promoter. (B) Transductional titers of lentiviral vectors encoding eGFP-fused TuDs with and without intervening WPRE determined by flow cytometric analysis of eGFP expression. (C) Dual-luciferase assay showing improved miRNA suppression activity of WPRE-fused TuDs targeting miR-16, -21, and -203 after lentiviral transduction using an MOI of 100 for all TuD-encoding lentiviral vectors. Included controls are similar for experiments performed with TuD16 and TuD21. Data are depicted as mean + SEM. Asterisks above columns show P-values for comparison with the negative control WPRE-MCS (asterisks in black) or MCS (asterisks in gray) cotransfected with psiCHECK-vector containing a perfect miRNA target site. TU, transducing units; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) not significant.

FIGURE 3.

Unchanged rates of nuclear RNA export by fusion of TuDs to WPRE. The nuclear export rate of TuD-encoding RNA evaluated as the ratio between cytoplasmic and total eGFP RNA levels after eGFP-specific RT-qPCR (A) or as the ratio between cytoplasmic and nuclear eGFP RNA levels after RNA-FISH analysis (D). (B) Example of RNA-FISH staining using eGFP-specific probes in HeLa cells transfected with 400 ng of plasmid containing WPRE-TuD16, TuD16, WPRE-MCS, or MCS. (C) Total, nuclear, and cytoplasmic eGFP RNA levels measured as pixel intensities after FISH staining. Data are depicted as mean + SEM. (*) P < 0.05, (**) P < 0.01, (ns) not significant.

FIGURE 4.

Colocalization between TuD-encoding RNA and P-bodies regardless of fusion of the TuDs to WPRE. (A) Combined RNA-FISH using eGFP-specific probes and immunostaining with antibodies targeting the P-body-specific marker hDcp1a shows colocalization between TuD-encoding RNA and P-bodies for WPRE-TuD7, WPRErev-TuD7, and TuD7 whereas no colocalization was observed for WPRE-MCS, WPRErev-MCS, and MCS after transient transfections of HeLa cells. Due to Alexa488 labeling of the secondary antibodies, eGFP protein and hDcp1a were visualized using the same filter. (B) Dual-luciferase assays evaluating levels of miR-21 suppression mediated by WPRE-TuD21 and TuD21 after knockdown of the P-body associated proteins Lsm4 and DDX6 using siRNA. Scrambled (scr) siRNAs and untransfected cells were used as controls. Data are depicted as mean + SEM. Asterisks above columns show P-values for comparison with the negative control WPRE-MCS cotransfected with psiCHECK-vector containing a perfect miRNA target site. Asterisks indicate significance as follows: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) not significant.

FIGURE 5.

Truncation of WPRE further enhances TuD function. (A) Schematic representation of WPRE-fused TuDs with deletion in WPRE (numbers refer to nucleotides of WPRE present or deleted according to GenBank accession number J04514). Evaluation of the impact of the X-protein promoter in WPRE (B), the hairpin-shaped part of the α-subunit (C), and the γ-, α-, and β-subunits of WPRE (D) on miRNA suppression activity of TuDs. Data are depicted as mean + SEM. Black asterisks above columns show P-values for comparison with the negative control WPRE-MCS cotransfected with psiCHECK-vector containing a perfect miRNA target site; gray asterisks above columns show P-values for comparison with WPRE-TuD cotransfected with psiCHECK-vector containing a perfect miRNA target site. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) not significant.