Preferential microRNA targeting revealed by in vivo competitive binding and differential Argonaute immunoprecipitation

Abstract

MicroRNAs (miRNAs) have been described to simultaneously inhibit hundreds of targets, albeit to a modest extent. It was recently proposed that there could exist more specific, exceptionally strong binding to a subgroup of targets. However, it is unknown, whether this is the case and how such targets can be identified. Using Argonaute2-ribonucleoprotein immunoprecipitation and in vivo competitive binding assays, we demonstrate for miRNAs-21, -199-3p and let-7 exceptional regulation of a subset of targets, which are characterized by preferential miRNA binding. We confirm this finding by analysis of independent quantitative proteome and transcriptome datasets obtained after miRNA silencing. Our data suggest that mammalian miRNA activity is guided by preferential binding of a small set of 3'-untranslated regions, thereby shaping a steep gradient of regulation between potential targets. Our approach can be applied for transcriptome-wide identification of such targets independently of the presence of seed complementary sequences or other predictors.

Figure 1.

Schematic representation of dual-fluorescent reporter assays. (A and B) Reporter constructs for the determination of miRNA activity in cells are cotransfected with two types of inhibitors: the effect of an miRNA on a target is assessed by cloning the UTR of interest downstream of GFP and treatment with small molecule inhibitors (LNA-antimiRs, A). Conversely, the inhibitory effect of a UTR on an miRNA can be assessed by cloning generic binding sites downstream of GFP and co-transfecting plasmids expressing the UTR of interest downstream of the non-interfering fluorophore iRFP (infrared fluorescent protein (36), B). (C) MiRNA activity is assessed by measuring the GFP normalized to the RFP (tdTomato) signal after reporter transfection into cells. (D) Readouts for GFP and RFP values for individual cells are obtained by automated microscopic image acquisition and high content image analysis. Selection of cells from a narrow range with little miRNA inhibition by the reporter reduces variability due to different transfection strength between cells.

Figure 2.

Superior regulation of CNTFR and TMEM2 3′-UTRs is mediated by strong miRNA binding to single target sites. (A) Predicted hybridization of the binding sites of selected targets. (Mutant and modified sites are depicted in Supplementary Figure S4B). (B) Results of the dual-fluorescent reporter assays. Measurements with the antimiRs LNA-21 (n = 8) or LNA-let-7 family (n = 5) were normalized to LNA-Ctrl. Depicted are values from cells in the bin with 100–200 AFU RFP values (as in Figure 1D). (C) Results of the competitive binding assay. Dual fluorescent reporters specific for miR-21 or let-7 activity (binding sites are depicted in Supplementary Figure S2) were co-transfected with the analyzed UTRs inserted 3′ of iRFP to compare their potencies as inhibitors of the respective miRNAs. Transfection ratios of reporter to inhibitor were 1:1 for miR-21 (n = 4) and 1:10 for let-7 (n = 7) targets. Depicted is the bin with 400–800 AFU for miR-21 and 100–200 AFU for let-7. (D and E) Design and results of the Ago2-RIP assay to compare changes in RIP-fraction upon seed mutation (n = 5). *P < 0.05 in ANOVA for repeated measures with a post-hoc Tukey’s test.

Figure 3.

Experimental design and distributions of mRNA fold changes upon miRNA inhibition. (A) Experimental design for simultaneous measurement of antimiR-mediated depletion from Ago2-complexes (RIP-enrichment changes) and mRNA expression changes for the four miRNAs (or miRNA families) in NIH3T3 fibroblasts. (B) Non-targets and conserved targets were grouped as indicated on the left. Panels below depict median mRNA fold changes ± bootstrap standard errors. For the contrasts P-values (*P < 0.05, **P < 0.01 in a Mann–Whitney U-Test) and the difference of medians of log₂ transformed data are indicated separated by a slash. N-numbers are indicated in parentheses.

Figure 4.

Transcriptome-wide analysis shows strong correlation between preferential regulation and preferential binding. (A) Boxplots of fold changes in RIP-enrichment (_ΔEnrichment) upon miRNA inhibition depicted separately for conserved targets and non-targets (no 7- or 8-mer seed match in the 3′-UTR). MRNAs were binned based on their expression fold changes (depicted as boxplots in the lower row). (B) Distributions of mRNA fold changes for the indicated conserved target and non-target groups as described in the main text. Panels below depict median mRNA fold changes with bootstrap standard errors. For group comparisons P-value (*P < 0.05, **P < 0.01 in a Mann–Whitney U-Test) and the difference of medians of log₂ transformed data are indicated separated by a slash. N-numbers are indicated in parentheses.

Figure 5.

Proteome and tissue analysis confirms preferential regulation of predicted preferential targets at endogenous miRNA levels. (A) Experimental design for SILAC proteomics analyses of miR-199–3p targets in NIH3T3 fibroblasts. (B) Protein fold changes for the indicated target and control groups after miR-199–3p overexpression (miR-mimic, left) or knockdown (antagomiR, right). Predicted top candidates, which were identified in the knockdown assay, are indicated in red. Top Ctrl. indicates targets with an mRNA fold change above and RIP-enrichment fold change below the threshold. (C) As in (B) for mRNA fold changes after introduction of miR-199–3p in neonatal mouse cardiac myocytes (28). (D) As in (B) for average mRNA fold changes between wild-type (wt) and miR-21 knockout mice, measured by RNA-seq in mammary gland (29) or microarray in blood neutrophils and macrophages (30). Averages were calculated after quantile normalization. Graphs for individual experiments depicted in Supplementary Figure S5. (E) As in (B) for protein fold changes following let-7b knockdown in HeLa cells, measured by pSILAC (31). N-numbers, P-values and median differences indicated as in Figure 4. ^#P < 0.05 for Top candidates versus Top Ctrl. and P < 0.01 versus all other groups.