Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression

Abstract

Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed.

Keywords: base pair complementarity; competing endogenous RNA; cooperatively; gene regulation; miRNA; miRNA degradation; target abundance.

Graphical abstract

Figure 1

Derepression of Target mRNAs Occurs at a High Threshold of Added Target Sites (A) Dual-color fluorescent reporter constructs containing zero (0s), one (1s), or three (3s) 8-nt miRNA site(s) in the 3′ UTR of mCherry. (B and C) ESCs transfected with either a 3s (B), 1s (C), or 0s reporter construct (n = 3) with miRNA-binding sites for miR-294, -293, -92, or -16. Mean mCherry fluorescence (B, left), and mCherry fluorescence normalized to the 0s control (B, right and C) across 20 bins of eYFP. (D–I) RNA-seq results (n = 2) of sorted ESCs shown in Figure S1A. ESCs were transfected with a 3s reporter for miR-293 (D–F) or miR-92 (G–I), or a 0s control, and gated for cells with low (eYFP_low) (D and G), intermediate (eYFP_int.) (E and H), or high eYFP (eYFP_high) (F and I) expression. Cumulative distribution function (CDF) of mRNA changes for predicted target genes with the indicated context+ score (cs+) bins (color) or for genes with no miRNA site (black). mCherry MREs per cell evaluated by qPCR are shown on each graph. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, one-sided Kolmogorov-Smirnov (K-S) test. Also see Figures S1B and S1C. (J) Relationship between reporter protein fluorescence measured by flow cytometry and RNA copies per cell evaluated by qPCR of ESCs transfected with the 0s reporter and sorted into four different bins of eYFP-expressing cells. Line represents non-linear regression of data points; respective equations are shown. (K) Protein fluorescence values shown in (B) transformed to RNA copies per cell using the equations shown in (J). Vertical lines represent the DRT (dotted lines) or IC₅₀ (solid lines). (L and M) Bar plot of DRT (L) and IC₅₀ (M) shown in (K). (N) Transcriptome TA_app of ESCs transfected with the 0s reporter and sorted for low eYFP-expressing cells (ESC 0 s eYFP_low). (O) Fractional contribution of the largest potential contributors to transcriptome TA_app of ESC 0s eYFP_low. Potential contributors were binned by their context+ score, and the top potential contributors are plotted within each bin. Data represent mean ± SEM for (B), (C), and (K).

Figure 2

Derepression Threshold Values Are Insensitive to Changes in miRNA Activity (A–F) ESCs co-transfected with a 3s reporter for miR-293 (A–D), miR-92 (E and F), or respective 0s reporter control, and different concentrations of Ant-293 (n = 3) (A and B), miR-293 (n = 6) (C and D), or miR-92 (n = 6) (E and F). (A, C, and E) Mean mCherry fluorescence (left), and mCherry fluorescence normalized to the 0s control (right) across 20 bins of eYFP. (B, D, and F) Protein fluorescence values shown in (A), (C), and (E) were transformed to RNA copies per cell using the equations shown in Figure 1J. Vertical, dotted lines denote the DRT. Data represent mean ± SEM for all panels.

Figure 3

Extensively Paired Sites Are More Potent Than 8-nt Sites and Trigger miRNA Decay (A) Schematic overview of the different AldoA-expressing adenovirus constructs. (B–G) Primary hepatocytes (n = 4) infected with different MOIs of Ad-AldoA 1s (B and C), bu4 (D–G), or respective Ad-AldoA Mut controls at either basal miR-122 levels (B–G) or with co-infected Ad-miR-122 (B and C). Relative levels of AldoA (B and D), miR-122 target genes and control non-target gene (ApoM) (C and E), or miR-122 (F). Vertical, dotted lines denote the DRT. miRNA levels are relative to the lowest MOI of Ad-AldoA Mut at basal miR-122 levels. (G) Northern blot analysis of miR-122, miR-16, and U6 at basal miR-122 levels. Data represent mean ± SEM for all panels. Also see Figure S7.

Figure 4

miRNA Target Derepression for let-7, miR-194, and miR-192 Also Occurs at a High Threshold of Added MREs (A) Absolute copies per cell of hepatocyte miRNAs. (B) Schematic overview of Ad-AldoA constructs harboring a mutated site (Mut), or one (1s) 8-nt binding site for let-7, miR-194, -192, or -101. (C–F) Primary hepatocytes infected with different MOIs of Ad-AldoA Mut or 1s (let-7). (C) Relative expression of let-7 target genes and control non-target gene (ApoM). (D–F) CDF of RNA-seq data (n = 2) showing mRNA changes for predicted target genes of let-7 with the indicated cs+ bins (color) or for transcripts with no miRNA site (black). (G–L) Hepatocytes infected with different MOIs of Ad-AldoA Mut, 1s (miR-192), or 1s (miR-194), in addition to MOI 15 Ad-miR-192/194. Relative levels of miR-194 (G) or miR-192 (H) target genes, and control non-target gene (ApoM) (I). CDF of RNA-seq data (n = 2) showing mRNA changes for predicted target genes of miR-194 (J and K) or miR-192 (L) with the indicated cs+ bins (color) or for genes with no miRNA site (black). AldoA MREs per cell evaluated by qPCR are shown on each graph. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, one-sided K-S test. Vertical, dotted lines denote the DRT. Data represent mean ± SEM (n = 4) for all panels.

Figure 5

The 6-, 7-, and 8-nt Sites Contribute Comparably to Target Abundance of miR-122 (A) Schematic overview of Ad-AldoA constructs used in this figure. (B–E) Primary hepatocytes infected with different MOIs of Ad-AldoA miR-122 8-mer, miR-122 7-mer-m8, miR-122 6-mer, or Mut. Absolute copy numbers per cell of AldoA (B), relative gene expression of GFP (C), and of miR-122 target genes or control non-target gene (ApoM) (D). (E) CDF of RNA-seq data (n = 2) showing mRNA changes for predicted target genes of miR-122 with the indicated cs+ bins (color) or for genes with no respective miRNA site (black). AldoA MREs per cell evaluated by qPCR are shown on each graph. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, one-sided K-S test. See also Figures S5C and S5D. Vertical, dotted lines denote the DRT. Data represent mean ± SEM (n = 4) for all panels.

Figure 6

Derepression Is Enhanced When Mediated by Closely Spaced MREs (A) Schematic overview of Ad-AldoA constructs used in this figure. (B–J) Primary hepatocytes infected with different MOIs of Ad-AldoA constructs shown in (A). Relative gene expression of let-7 target genes (B, E, and H), miR-122 target genes (C and F), or control non-target gene (ApoM) (D, G, and I). (J) CDF of RNA-seq results (n = 2) showing mRNA changes from hepatocytes infected with MOI 80 of Ad-AldoA let-7 +58nt or 2x +58nt for predicted target genes of let-7 (with no predicted target sites for miR-122) with the indicated cs+ bins (color) or for genes with no let-7 or miR-122 miRNA sites (black). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, one-sided K-S test. (K–N) Hepatocytes infected with different MOIs of Ad-AldoA Mut, miR-122, let-7, or 2x +58nt. Relative gene expression of predicted target genes for both let-7 and miR-122 (K), let-7 target genes (L), a miR-122 target gene (M), or a control non-target gene (ApoM) (N). Vertical, dotted lines denote the DRT. Data represent mean ± SEM (n = 4) for all panels.

Figure 7

Mathematical Simulation of the Mixed-Affinity Model (A) Simulated effects of changing miR-293 concentrations in ESCs on 8-nt target site repression (as performed in Figure 2), using the mathematical framework of the mixed-affinity model. Simulated mCherry fold-changes of the 1s reporter normalized to the 0s control are either plotted against mCherry (left) or eYFP (middle), indicating the IC₅₀ (solid lines) and DRT (dashed lines), for each of the three simulated miR-293 levels (in copies per cell [cpc]). Also plotted is the binding affinity distribution of all simulated target sites (right), with the K_D of each site normalized to that of an 8-nt site and the abundance of each site scaled by its normalized K_D. Abundance of 8-, 7-, and 6-nt, and low-affinity sites for miR-293 are plotted separately (purple, blue, cyan, and gray, respectively). The abundance of the canonical sites was determined by sequencing and that of the low-affinity sites was set such that the IC₅₀ matched that observed in Figure 1C. (B) Site occupancy for the simulations in (A). Plotted is the simulated free fraction of mCherry 1s reporter as a function of its expression, otherwise as in (A). (C) Simulated effects of changing miR-293 concentrations in ESCs on 8-nt target site occupancy using the mathematical models of site competition from Bosson et al. (2014) (left) or Jens and Rajewsky (2015) (right). Simulated free fraction of an added 8-nt target site is plotted as a function of its expression as in (B), using the binding-affinity distributions of the simulated target sites of the original studies, plotted as in (A). The IC₅₀ inferred from Figure 1C is indicated (dotted lines). (D) Simulations using the models of (C) but adding low-affinity sites to alleviate the discrepancy between the simulated and experimental results, otherwise as in (C).