Retargeting target-directed microRNA-decay sites to highly expressed viral or cellular miRNAs

Abstract

MicroRNAs (miRNAs) are pervasive regulators of gene expression, necessitating the development of tools to inhibit individual miRNAs for functional studies or therapeutic targeting. Specialized base-pairing configurations between a miRNA and an RNA target site can trigger the degradation of the targeting miRNA through target-directed miRNA decay (TDMD). Previous work has identified several natural sites that induce TDMD of specific miRNAs. We explored retargeting known TDMD sites for the inhibition of heterologous miRNAs, including several encoded by Kaposi's Sarcoma-associated herpesvirus (KSHV). We focused particularly on miR-K11, a viral mimic of the oncogenic miRNA miR-155. miRNA pairing architectures based on the TDMD site in the long non-coding RNA Cyrano outperformed other retargeted sites. Cyrano-like inhibitors were specific for viral miR-K11 over cellular miR-155 and vice versa. Lentiviral delivery of a Cyrano-like miR-K11 inhibitor into KSHV-transformed primary effusion lymphoma (PEL) cells impaired their viability, showing that miR-K11 promotes KSHV-dependent PEL cell survival. Surprisingly, inactivation of ZSWIM8, a key mediator of TDMD, did not substantially affect miRNA inhibition by retargeted Cyrano-based inhibitors in 293T or PEL cells. Together, our results demonstrate the feasibility of retargeting natural TDMD sites to highly expressed viral or cellular miRNAs and further define features of effective encoded miRNA inhibitors.

Graphical Abstract

Figure 1.

Assessment of retargeted TDMD sites in miRNA luciferase sensor assays. (A) Validated natural TDMD sites chosen for retargeting. The site in UL144-145 can bind to either miR-17 or miR-20, which differ from each other at nts 1 and 12. See Supplementary Figure S1 for all retargeted sites. (B) Vectors used to assess the efficiency of retargeted TDMD sites in miRNA luciferase sensor assays. pLCE was used to express single TDMD sites (left) or the targeting miRNAs (middle). pLCG was used as a miRNA sensor through three completely complementary sites, resulting in Ago2-mediated cleavage. The corresponding empty control vectors used for normalization are shown in the bottom row. pLCR, used to express Renilla Luciferase in each well, is not shown.

Figure 2.

Retargeting TDMD sites to viral and human miRNAs. (A) Results for miR-K11 using a low amount of miRNA expression plasmid (2.5 ng per 24-well) to distinguish effective and non-effective sites. Firefly luciferase sensor results were normalized to an internal Renilla luciferase transfection control (pLCR), a no TDMD-site empty vector control (pLCE), a no miRNA empty vector control (pLCE), and a firefly luciferase vector without target sites (pLCG). (B) As in (A) but showing results for miR-122. (C-F) As in (A) but the amount of transfected miRNA expression plasmid was increased to 10 ng due to relatively lower miRNA activity for (C) miR-155, (D) miR-K3, (E) miR-K1 and (F) miR-K4-3p. Error bars represent mean ± SD. Unless indicated by a horizontal bar, significance refers to comparisons against the no TDMD-site EV control (none) and was calculated using unpaired two-tailed Student's t tests. n = 3 independent experiments. * indicates P < 0.05. ns not significant.

Figure 3.

Changes in the central bulge identify an optimized Cyrano-like TDMD site. (A) Schematic of the changes introduced into the bulge configuration of the miR-K11-retargeted Cyrano TDMD site. See Supplementary Figure S3 and Supplementary Table S1 for other miRNA target sites. (B–D) results of luciferase assays as in Figure 2, for (B) miR-K11, (C) miR-122 and (D) miR-K4-3p, using a higher amount of miRNA than for Figure 1 (i.e. 10ng per 24 well), to achieve more robust sensor knock-down and higher resolution between effective TDMD sites. (E–G) Additional results for miR-K3, miR-K1, and miR-155, comparing only the sponge site, the original Cyrano site, and the Cyrano-C site. Error bars show mean ± SD. Significance refers to comparisons against single sponge inhibitors, unless specified by horizontal bars for other comparisons, and was calculated using unpaired two-tailed Student's t tests. n = 3–6. * indicates P < 0.05. ns: not significant.

Figure 4.

Improved miRNA inhibition through optimized spacing and increased number of Cyrano-like sites. (A) Schematic explaining the spacings between two Cyrano-like type C sites used in panel B. Two Cyrano-like type C sites were separated by 7, 14 and 60 sites or incorporated into a TuD secondary structure. (B) miR-K11 activity sensor assays to determine the benefit of using two miRNA inhibitory sites, as outlined in panel A. Significance refers to comparisons specified by horizontal bars and was calculated using unpaired two-tailed Student's t tests. n = 3–6. Data are represented as mean ± SD. *P < 0.05. ns: not significant. (C) Additional significance calculations from data in panel B. (D) Schematic illustrating miRNA inhibitors used for panel E. The number of Cyrano-like type C sites was increased to 2, 4, 8 and 12, with 14 nts spacers. (E) miR-K11 activity sensor assays to determine the benefit of using additional miRNA-inhibitory sites. Cyrano-C pairing improved miRNA repression compared to equally spaced sponge sites at 1, 2 and 4 sites. All Cyrano-C and sponge configurations resulted in significant sensor derepression. Seed match scrambled miR-K11 Cyrano-C sites did not confer robust sensor derepression. Significance refers to comparisons specified by horizontal bars and was calculated using unpaired two-tailed Student's t tests. n = 3. Data are represented as mean ± SD. *P < 0.05. ns not significant.

Figure 5.

Cyrano-like miRNA inhibitors distinguish miRNAs with shared seed sequences. (A) Comparison of human miR-155 and its KSHV mimic miR-K11. Identical seed sequences (nts 2–8) are in red, other shared nts are in purple. (B) Predicted base pairing of miR-155 (top) or miR-K11 (bottom) to the Cyrano-like miRNA inhibitors (type C) used in panels C–D. (C) miR-155 luciferase activity sensor assay shows specificity of the miR-155 inhibitor for miR-155. (D) as for panel C, but for miR-K11. In panels C–D, significance refers to comparisons against the control vector without miRNA inhibitory sites, unless specified by horizontal bars, and was calculated using unpaired two-tailed Student's t tests. n = 3. Data are represented as mean ± SD. *P < 0.05. ns: not significant.

Figure 6.

ZSWIM8 is dispensable for miRNA inhibition by retargeted TDMD sites. (A) Western blot analysis of 293T ZSWIM8 KO cell pools following Cas9-mediated gene editing. (B) Endogenous miR-7 activity was increased in ZSWIM8 KO cell pools compared to AAVS1, as measured by miR-7 specific activity sensors. (C) No change in miR-K11 activity was observed in ZSWIM8 KO 293T cells compared to sgAAVS1 control pools following transfection with miR-K11 expression plasmids, miR-K11 inhibitory plasmids (sponge or Cyrano-C), miRNA sensors, and internal control. Significance values show unpaired two-tailed Student's t test. n = 3–4. Data are represented as mean ± SD. *P < 0.05.

Figure 7.

miR-K11 promotes the growth of the PEL cell line BCBL-1. (A) Schematic of the lentiviral expression cassette for miRNA inhibitors in this figure. The inhibitors used were the 12 × Cyrano-C sites for miR-K11 (12 × miR-K11), the 12× sponge sites for miR-K11 (12 × sponge), the 12 × Cyrano C sites for miR-122 (12 × miR-122), with matched spacing. (B) TaqMan qRT-PCR analysis of miR-K11 expression relative to RNU48 in the KSHV-transformed PEL cell line BCBL-1, two days after lentiviral transduction at a MOI of 5. (C) Cumulative growth curves of BCBL-1 cells transduced with lentiviruses encoding no miRNA inhibitory sites (none), 12× miR-K11 Cyrano-C, 12× miR-K11 sponge or 12× miR-122-directed Cyrano-C inhibitors at an MOI of 5. (D) Cumulative growth curves as in (B), but in BJAB cells, a non-KSHV infected control. Significance values were calculated using an unpaired two-tailed Student's t-test. n = 3–10. Data are represented as mean ± SD. *P < 0.05. ns: not significant.

Figure 8.

ZSWIM8 knockout in BCBL-1 does not rescue miR-K11 inhibitor lethality. (A) Western blot analysis of ZSWIM8 expression in sgRNA-transduced BCBL-1/Dox-inducible-Cas9 cell pools following transient induction of Cas9 expression. (B) End point of cumulative growth curves on day 8 after miRNA inhibitor transduction of the cell pools from panel A. Transductions with either an empty vector (none) or a 12× miR-K11 Cyrano-C inhibitor were performed at MOI of 5. n = 3. Significance values show unpaired two-tailed Student's t test. Data are represented as mean ± SD. *P < 0.05.