Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds

Abstract

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

Keywords: Dicer-independent shRNA; Drosha; Pol-II driven miRNA scaffold; RNAi; U1 promoter; agoshRNA; agshRNA; miR-324; miR-451; passenger strand activity.

Figure 1

Comparison of Dicer-Independent shRNA Designs and Identification of Potent agshRNAs Targeting VEGF (A) Predicted secondary structure of a previously reported shRNA targeting site 9 in the VEGF gene, based on a conventional sense-loop-antisense design (sh9, left side); Ago2-dependent designs, based on the 19/5 agoshRNA structure, as reported by the B. Berkhout lab (agosh9, middle part); or the agshRNA design, as described by the Yen group (agsh9, right side). Arrows indicate predicted Dicer and Ago2 cleavage sites, and position 2-7 of the guide RNA (seed site) is shown with braces. The guide and passenger strands are depicted in green and red, respectively. The frequently used Brummelkamp loop is shown in blue. The invariant unpaired A-C fork, as also present in miR-451, is highlighted in purple. (B) Schematic diagram of the psiCHECK2-based reporter encoding a Renilla luciferase-mVEGF cDNA fusion transcript (used for screening purposes) and the dedicated T12-sense or T12-antisense reporters (used for testing guide and passenger strand activity, respectively), which are used in combination with the various U6-driven shRNA expression plasmids. Name (T9, target site 9) and location (484, 3′ end of target site in the cDNA) of six chosen targets are indicated at the top. The illustration at the bottom shows the 105-nt-long cDNA region of mVEGF (dubbed T12) encompassing site 12, which was cloned into the psiCHECK2-based reporter shown above in sense or antisense orientation (numbers indicate cDNA position). (C) Predicted secondary structure of the conventional sense-loop-antisense sh12.3 (left side) and the Ago2-dependent agsh12.3 (right side) targeting site 12.3 in VEGF. Arrows and colors as indicated in (A). (D) Knockdown activity of agshRNAs designed to target the mVEGF sites, estimated by co-transfection with the mVEGF-fused reporter, as illustrated in the top part of (B). Renilla luciferase (Rluc) and Firefly luciferase (Fluc) activities were measured in relative units of light (RLU). The Rluc:Fluc ratio was normalized to the empty control (black bar) and plotted as the mean of three replicates plus SDs. An irrelevant agshIRR was included as a non-targeting control (gray bar). (E) Guide and passenger strand activity for agsh12.3 and a conventionally designed shRNA predicted to target site 12.3 using the dedicated T12-sense and T12-antisense reporters, respectively (see B). The Rluc:Fluc ratio was normalized to the empty control and plotted as the mean of three replicates plus SDs. (F) Northern blot analysis of small RNA from transfected HEK293 cells using probes detecting guide strand RNA (antisense probe, autoradiogram on the left side) or passenger strand RNA (sense probe, autoradiogram on the right side). Size of 20-, 30-, and 40-nt bands of the RNA decade marker is indicated, and agsh12.3- and sh12.3-specific bands are marked by arrows and labeled a–g. A cropped image with detection of the native U6 snRNA band (loading control) is shown below. (G) Diagram indicating probe specificity. Antisense probe, light green; sense probe, light red; Fluc, Firefly luciferase; HSV-tk, Herpes simplex virus thymidine kinase promoter; mVEGF, murine vascular endothelial growth factor; pA, polyadenylation signal; Rluc, Renilla luciferase; shRNA, short hairpin RNA; SV40, simian virus 40 promoter; T6, T-rich Pol III termination signal; U6, human U6 snRNA promoter.

Figure 2

Potent Knockdown of VEGF Using Dicer-Independent shRNAs Expressed as Pol II-Driven pri-miR-324 or -451 Transcripts (A) Diagram of the miR451-agsh12.3 chimera showing the sequence and predicted stem loop structure around the Drosha cleavage site. (B) Knockdown activity of U1-expressed agshRNA embedded in various miRNA scaffolds, estimated by co-transfection with psiCHECK-mVEGF-T12-sense. The Rluc:Fluc ratio was normalized to the empty control (black bar) and plotted as the mean of three replicates plus SDs. Endogenous miR-30a and miR-451 were included as non-targeting controls (gray bars). Exact compositions of the miRNA-agshRNA chimeras at the central stem loop structure are shown in Figure S2. (C) Passenger strand activity as measured using the psiCHECK-mVEGF-T12-antisense reporter and plotted as described in (B). (D) Northern blot analysis of small RNA from transfected 293T cells using probes detecting guide strand RNA (antisense probe, autoradiogram on the left side) or passenger strand RNA (sense probe, autoradiogram on the right side). Size of 20-, 30-, and 40-nt bands of the RNA decade marker is indicated, and agsh12.3-specific bands are marked by arrows and labeled a–c. A cropped image with detection of the native U6 snRNA band (loading control) is shown below. (E) Knockdown of intracellular (lysate) and secreted (media) mVEGF by U1-expressed miR451-agsh12.3, estimated by co-transfection with an mVEGF-expressing plasmid and western blot analysis. Total protein levels in each lane after membrane transfer (relative values) are shown below, and lines and arrows indicate protein size markers and the position of the mVEGF band, respectively. Endogenous miR-451 and U6-driven agsh12.3 were included as a non-targeting and a positive control, respectively.

Figure 3

Knockdown Activity of miRNA-agshRNAs Is Dicer Independent (A) Knockdown levels of U6-driven constructs in Dicer-knockout cells (NoDice 2-20, purple bars) and the parental 293T control cells (gray bars), as based on psiCHECK-mVEGF-T12-sense reporter activity. The Rluc:Fluc ratio was normalized to the empty control and plotted as the mean of three replicates plus SDs. An irrelevant agshIRR was included as a non-targeting control. For the hatched bars, the agshRNA or shRNA dose was lowered from 34 to 0.5 ng. p values for selected statistical tests are indicated. (B) Knockdown activity of U1-driven miR324- and miR451-agsh12.3 in Dicer-knockout cells (purple bars) and the parental 293T control cells (gray bars) measured and plotted as described in (A). Endogenous miR-30a and miR-451 were included as negative controls. (C) Northern blot analysis of small RNA from transfected DcrKO 293T cells (NoDice 2-20) using probes detecting guide strand RNA (antisense probe, autoradiogram on the left side) or passenger strand RNA (sense probe, autoradiogram on the right side). Arrows indicate RNA size markers for the 20-, 30-, and 40-nt bands, and agsh12.3-specific bands are marked by arrows and labeled a–c. A cropped image with detection of the native U6 snRNA band (loading control) is shown below.

Figure 4

Easy Design and Efficient Targeting of a Gene of Choice (A) Knockdown activity of U1-driven agshRNA targeted against the human RRM2 gene (site 887, dark gray; site 1354, light gray;) when embedded in miR-215, -324, -409, or -451 scaffolds. For comparison, data from Figure 2B have been replicated (VEGF targeting shown in green). The Rluc:Fluc ratio, as measured by dedicated psiCHECK-based reporters, was normalized to the empty control and plotted as the mean of three replicates plus SDs. Endogenous miR-30a and miR-451 were included as negative controls. (B) Schematic diagram of the CMV and U1 Pol II expression cassettes used to generate miRNA-agshRNA transcripts. CMV transcripts terminate using the bovine growth hormone polyadenylation signal (denoted pA), while the U1 snRNA transcripts stop using an U1 terminator box (denoted U1t). (C) Diagram depicting the simplicity of reconfiguring an existing conventional shRNA (here illustrated for the HIV-1 site 1-targeting shRNA denoted shS1) into a miR324-agshRNA scaffold for Pol II expression. The 21-mer guide strand for a given target forms the 5′ side and the loop (highlighted in green), while complementary sequences constitute the 3′ side (shown in yellow). The invariant unpaired A-C fork (highlighted in purple), present in the agshRNA design shown in Figure 1A, is simply kept and placed at the natural miR-324 Drosha cleavage site. (D) Knockdown activity of U1- (dark blue) or CMV- (light blue) expressed agshRNA targeted against the HIV-1 site 1, from miR-324 or -451 scaffolds, measured and plotted as described in (A).