Properties and kinetics of microRNA regulation through canonical seed sites

Abstract

MicroRNAs are a fundamental class of small RNAs involved in post-transcriptional gene regulation; however, the mechanism by which microRNAs regulate their gene targets in animals remains poorly understood. Practically, a mechanistic understanding of microRNA binding and regulation is crucial for the rational design of microRNA-based vectors for RNA interference. In this report, we focus on the largest known class of microRNA targets, the canonical seed targets, and explore the factors involved in modulating target downregulation in vivo at the protein level. Using an in vivo sensor assay in the ascidian Ciona intestinalis, we quantify miR-124-mediated downregulation of 38 canonical seed targets cloned from the Ciona genome as well as 10 control non-targets. Supporting previous findings, we observed that the seed type and number of seed sites are correlated with downregulation. However, up to a 50% variation in downregulation levels was observed for targets within the same seed class, indicating a role of non-seed factors in modulating downregulation. Although we did not observe a significant correlation of previously reported non-seed determinants with downregulation levels at saturation in our assay, our data suggest that two previously identified factors, secondary structure and 3'end complementarity, may play a role in the initial kinetics of microRNA-target binding. Importantly, using different concentrations of miR-124 we show that dose-dependent target downregulation profiles follow Michaelis-Menten kinetics. In summary, our findings emphasize the importance of non-seed factors as well as the importance of cellular concentrations of microRNAs relative to their targets when studying the mechanisms of endogenous microRNA regulation.

Keywords: Michaelis-Menten; canonical target; miRNA targets; microRNA; seed site.

Figure 1.

Quantitative measurement of miRNA-mediated downregulation using in vivo reporter assay in Ciona intestinalis. (A) Downregulation of a target RFP construct relative to an control SV40 3’UTR CFP construct is measured in the presence (miR+) versus absence (control) of a targeting miRNA. For control embryos, a neutral YFP transgene was electroporated in place of the miR-124-expressing transgene as a mass control. (B) A representative pair of embryos for our in vivo reporter assay, where miR-124 is used as our targeting miRNA. RFP, CFP and Nomarski / Differential interference contrast (DIC) images are shown. (C) Downregulation (mean ± SD) for all 38 targets and 10 non-targets tested, categorized according to seed type. * indicates targets that were tested in our previous report (Chen et al, 2011), but are used in this report for quantitative analysis. We show all targets here to highlight the variation in percent downregulation for targets of the same seed type. (AS) indicates genes for which at least two alternative 3’UTR splice isoforms were tested, with at least one target and one non-target isoform tested. The blue bar for KH.C6.55.v7 with the associated blue axis range indicates that this non-target was upregulated instead of downregulated in our assay, which was consistent among three independent replicates.

Figure 2.

Presence of a canonical seed site has a significant effect on target downregulation. We tested if the presence of 7mer-A1, 7mer-m8, 8mer or multiple canonical seed sites was sufficient for significant target downregulation. *p<0.05, t-test with correction for multiple testing.

Figure 3.

miRNA target titration curves follow Michaelis-Menten kinetics. (A) miR-124 titration curves for Macho-, Macho-1-SeedKO1, Macho-1-SeedKO2 and LRP6. (B) miR-124 titration curves for HH, Neur, Hes1, PTBP1 and C13.2. (C) Michaelis-Menten kinetic constants V_max and K_m for each of the tested targets. (D) For each of the tested single-site targets in (A-C), the Pearson correlation of previously reported non-seed determinants (Grimson et al, 2007; Kertesz et al, 2007; Nielsen et al, 2007) is graphed as a function of downregulation at the specified miRNA-to-target concentration ratios or as a function of initial reaction rates (V_max/K_m) as determined by the respective Michaelis-Menten kinetic curves. Error bars show 95% confidence intervals for the Pearson correlation.