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Regulation of microRNA-mediated gene silencing by microRNA precursors

Nature Structural & Molecular Biology volume 21, pages 825–832 (2014)Cite this article

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Abstract

Processing of microRNAs (miRNAs) from their precursors to their biologically active mature forms is regulated during development and cancer. We show that mouse pri- or pre-miR-151 can bind to and compete with mature miR-151-5p and miR-151-3p for binding sites contained within the complementary regions of the E2f6 mRNA 3′ untranslated region (UTR). E2f6 mRNA levels were directly regulated by pri- or pre-miR-151. Conversely, miR-151–mediated repression of ARHGDIA mRNA was dependent on the level of mature miR-151 because only the mature miRNA binds the 3′ UTR. Thus, processing of miR-151 can have different effects on separate mRNA targets within a cell. A bioinformatics pipeline revealed additional candidate regions where precursor miRNAs can compete with their mature miRNA counterparts. We validated this experimentally for miR-124 and the SNAI2 3′ UTR. Hence, miRNA precursors can serve as post-transcriptional regulators of miRNA activity and are not mere biogenesis intermediates.

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Figure 1: miR-151-5p cleaves mouse E2f6 in the absence of a seed match.
Figure 2: miR-151-3p suppresses E2f6 expression by binding to the E2f6 3′ UTR adjacent to where miR-151-5p binds.
Figure 3: Precursor miR-151 competes with the mature miR-151-5p for binding to the E2f6 3′ UTR.
Figure 4: Pre-miR-151 binds to E2f6 in vivo and may protect the E2f6 transcript in quiescent tissues.
Figure 5: Regulation of miRNA activity by precursor miRNA is target specific.
Figure 6: Predicted widespread regulation of miRNA-mediated gene silencing by precursor miRNAs.

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Acknowledgements

We thank members of the Kay laboratory, in particular, L. Lisowski for help with fluorescence-activated cell sorting, F. Zhang for mouse dissection and culture of Dicer-knockout cells, L. Sobkowiak for RNA extraction from mouse tissues and S. Gu and H.K. Kim for critical comments and helpful discussion. We also thank J. Sage and T. Rando (both at Stanford University) for providing reagents and A. Lund (University of Copenhagen) for advice on the biotinylated pre-miR-151 pulldown assay. We obtained the catalytically inactive form of Ago2 (Ago2-D597A) as a kind gift from G. Hannon (Cold Spring Harbor Laboratory). This work was supported by the US National Institutes of Health (grant DK078424 to M.A.K.). P.N.V. is supported by a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research.

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Authors and Affiliations

  1. Department of Pediatrics, Stanford University, Stanford, California, USA

    Biswajoy Roy-Chaudhuri, Paul N Valdmanis, Yue Zhang, Qing Wang, Qing-Jun Luo & Mark A Kay

  2. Department of Genetics, Stanford University, Stanford, California, USA

    Biswajoy Roy-Chaudhuri, Paul N Valdmanis, Yue Zhang, Qing Wang, Qing-Jun Luo & Mark A Kay

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  1. Biswajoy Roy-Chaudhuri
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Contributions

B.R.-C., P.N.V. and M.A.K. designed the experiments. B.R.-C. performed the experiments with the exception of the ChIRP experiments, which were performed by Q.W. and Q.-J.L. Y.Z. performed bioinformatic predictions; B.R.-C., P.N.V. and M.A.K. analyzed and discussed the results and wrote the paper.

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Correspondence to Mark A Kay.

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Integrated supplementary information

Supplementary Figure 1 Overexpression and processing of miRNAs from plasmids.

Sequences of the mature mouse miRNAs are indicated in bold. (a) Northern blot analysis of miR-151-5p and miR-151-3p overexpressed specifically from their respective U6 promoter driven small hairpin (sh) constructs. (b) Northern blot analysis of miR-124 overexpressed from a sh construct. (c) Northern blot analysis of miR-124 overexpressed from a Pol II based CMV driven construct, pEZX-124.

Supplementary Figure 2 Pre-miR-151 antagonizes mature miR-151 binding to E2f6 3′UTR.

(a) Multiple alignments of the binding site of the pre-miR-151 within the E2F6 3′UTR region (top panel) and pre-miR-151 (bottom panel) across various species. The pre-miR-151 is color coded to show the 5p arm (brown) and the 3p arm (green). (b) Schematic of the base pairing of the A-to-I edited pre-miR-151 with the mouse E2f6 3′UTR. Positions of the two inosines are shown in purple. The additional A:I base pairing created by editing is indicated by a dot. (c) Northern blot analysis of miR-151 from the Pol II based pEZX-151 construct. The blot was probed with a LNA probe against miR-151-5p. (d) Dual-luciferase reporter assay with E2f6 3′UTR (WT), 3p del or 3p-5p swap constructs and Sh-151-5p or Sh-151-3p. (e) Dose-dependent dual-luciferase reporter assay with E2f6 3′UTR WT and modified targets in presence of increasing dose of only mature miR-151-5p (Sh-151-5p) or both pre-miR-151 and mature miR-151-5p (pEZX-151 or pEZX-DM). (f) Dual luciferase reporter assay with E2f6 3′UTR in presence of Sh or pEZX-constructs and DNA/LNA mixmer blocker complimentary to the site of E2f6 where miR-151-3p binds. (g) Northern analysis of processing of pre-miR-151 in Dicer knockout cells. For comparison, processing of pre-miR-151 from MEF cells is also shown. U6 serves as a loading control. (h) Dual luciferase reporter assay with E2f6 3′UTR in presence of a synthetic duplex miR-151 and increasing amounts of the (A to G substituted) form of pre-miR-151 (pEZX-DM) in Dicer KO cells Normalization was done with respect to duplex synthetic miR-122. As a control, pEZX-124 was used as shown. (d — f, h) For reporter assays, error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Supplementary Figure 3 miR-151-3p–binding site in E2f6 3′ UTR is not antagonistic or does not interfere with miR-151-5p binding to its site.

(a) Dual-luciferase reporter assay for E2f6 3′UTR construct in presence of increasing proportion of Sh-151-5p relative to either a dose of Sh-151-3p or a control Sh-S. (b) Dual-luciferase assay reporter assay for E2f6 3′UTR or E2f6-3p del (E2f6 3′UTR lacking miR-151-3p binding site) construct in presence of increasing dosage of Sh-151-5p. In each transfection, a fixed amount of Sh-151-3p is also transfected. (a, b) For reporter assays, error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Supplementary Figure 4 Pre-miR-151 binds to E2f6 3′ UTR in vitro.

Quantitative PCR analysis of E2f6 from a streptavidin pull-down of a biotinylated 4tU-containing synthetic pre-miR-151 RNA (I) oligo. Error bars, s.e.m. (n = 2 biological replicates, each with 3 technical replicates).

Supplementary Figure 5 Predicted widespread gene regulation by pre-miRNAs in competition with mature miRNAs for the same target.

(a) A flowchart of the bioinformatics pipeline to predict genome-wide interactions of the precursor miRNAs with their predicted target 3′UTR regions common to human and mouse that would overlap with the binding of their corresponding mature miRNA forms. (b) Dual-luciferase reporter assay for wild-type SNAI2 3′UTR or its modified forms (SNAI2 ran or SNAI2 swap) in presence of mature miR-124 (Sh-124). Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Supplementary Figure 6 Original images of gels and blots used in this study.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 2 and 4–6 (PDF 1405 kb)

Supplementary Table 1

Genome-wide predictions of competitions between mature miRNAs and their precursors to bind to overlapping targets (XLSX 233 kb)

Supplementary Table 3

List of targets predicted to be regulated by precursors of repeat encoded miRNAs, miR-28 and miR-340, in humans (XLSX 127 kb)

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Roy-Chaudhuri, B., Valdmanis, P., Zhang, Y. et al. Regulation of microRNA-mediated gene silencing by microRNA precursors. Nat Struct Mol Biol 21, 825–832 (2014). https://doi.org/10.1038/nsmb.2862

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