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

摘要

class="head no_bottom_margin" id="sec1title">IntroductionMicroRNA (miRNA) levels have long been known to influence the magnitude of target-gene repression (). More recent studies point out that the number of predicted binding sites present in the transcriptome also affects the activity of miRNAs (, ). Consistent with this concept, strong overexpression of natural or artificial RNAs that contain miRNA sites can titrate miRNAs away from natural targets, thereby reducing the repression of these transcripts (, , , , ). These observations are extended by the notion that a site-containing transcript found naturally within cells can act as competing endogenous RNA (ceRNA) and regulate other site-containing transcripts by increasing or decreasing the miRNA activity (, , , ).The ceRNA hypothesis remains controversial due to the lack of a plausible explanation for how modulating the expression of a single endogenous gene could perceptibly influence miRNA activity across all of its target sites. Two recent studies have empirically assessed the ceRNA hypothesis by quantifying the number of miRNA response elements (MREs) that must be added to detect ceRNA-mediated gene regulation (, ). Both studies agree that determining the number of transcriptomic miRNA-binding sites is crucial for evaluating the potential for ceRNA regulation and that miRNA-binding sites are generally higher than the number of miRNA molecules. However, they differ in two aspects: (1) the experimental approaches used to determine the number of “effective” transcriptomic miRNA-binding sites and (2) the impact miRNA concentrations have on the number of binding sites that must be added to detect target gene derepression (derepression threshold [DRT]).The discrepancies between these studies lead to different conclusions with respect to the likelihood of observing ceRNA effects in natural settings. The first study concluded that changes in ceRNAs must approach a miRNA’s target abundance before they can exert a detectable effect on gene regulation (). Furthermore, because target abundance for a typical miRNA is very high, regulation of gene expression by ceRNAs is unlikely to occur in differentiated cells under physiological settings or most disease settings (href="#bib11" rid="bib11" class=" bibr popnode">Denzler et al., 2014). In addition, the study shows that the DRT remains constant when miRNA activity is reduced. A subsequent review presents a mathematical model that assesses binding-site occupancy and competition at different assumed target abundances (href="#bib22" rid="bib22" class=" bibr popnode">Jens and Rajewsky, 2015). This in silico model predicts that only global and collective changes in binding sites can produce an effect on target abundance large enough to detectably derepress target genes, which concurs with the results and conclusions of href="#bib11" rid="bib11" class=" bibr popnode">Denzler et al. (2014).The second study presents a “hierarchical affinity model,” in which the miRNA abundance is proposed to determine the respective susceptibility to ceRNA-mediated regulation (href="#bib6" rid="bib6" class=" bibr popnode">Bosson et al., 2014). In this model, the suggestion is that, as miRNA concentration increases and Ago-miRNA complexes spread to weaker and weaker sites (with affinity inferred from the site hierarchy of 8-nt > 7-nt > 6-nt site), the effective target-site abundance grows too large for physiological ranges of ceRNA expression to influence repression. By this reasoning, physiological ceRNA changes can nevertheless influence repression by a more modestly expressed miRNA, with its correspondingly lower effective target-site abundance. Moreover, the use of high-throughput cross-linking to detect targets leads to lower target-abundance estimates, which further increases the plausibility of ceRNA regulation (href="#bib6" rid="bib6" class=" bibr popnode">Bosson et al., 2014). However, experimental support for the proposed influence of miRNA concentration is correlative and lacks direct experimental evidence, such as manipulation of miRNA activity and measurement of resulting DRT changes.href="#bib11" rid="bib11" class=" bibr popnode">Denzler et al. (2014) propose that sites of all different affinities contribute to the effective target abundance, regardless of the miRNA concentration. Here, we call the model of href="#bib11" rid="bib11" class=" bibr popnode">Denzler et al. (2014) the “mixed-affinity model” to distinguish it from the hierarchical affinity model. The mixed-affinity model recognizes that a high-affinity site will contribute more to effective target-site abundance than a low-affinity site (href="#bib11" rid="bib11" class=" bibr popnode">Denzler et al., 2014). However, in aggregate, low-affinity sites, because of their high numbers within the transcriptome, still make a substantial contribution to the effective target-site abundance for each miRNA—even for more modestly expressed miRNAs.Other studies suggest that the ceRNA crosstalk of two transcripts is stronger and more specific when they share a large number of sites to different miRNA seed families. This hypothesis emerged from observations in cancer models, in which the expression of a particular oncogene correlates with its pseudogene, and both transcripts share a high sequence homology in their 3′ UTRs and are reported to co-regulate each other through a ceRNA mechanism (href="#bib28" rid="bib28" class=" bibr popnode">Poliseno et al., 2010, href="#bib23" rid="bib23" class=" bibr popnode">Karreth et al., 2015). Even if transcripts containing multiple sites can exert an additive effect of independently acting binding sites, sites for each miRNA family would still have to individually reach the high thresholds necessary to observe target-gene derepression. Therefore the simple presence of multiple binding sites alone would not be expected to be sufficient to increase the likelihood of a ceRNA effect, unless the sites acted through a cooperative mechanism. Although the effect of cooperativity has been studied in the context of target-gene repression (href="#bib12" rid="bib12" class=" bibr popnode">Doench et al., 2003, href="#bib18" rid="bib18" class=" bibr popnode">Grimson et al., 2007, href="#bib30" rid="bib30" class=" bibr popnode">Saetrom et al., 2007, href="#bib7" rid="bib7" class=" bibr popnode">Broderick et al., 2011), it is unclear whether closely spaced miRNA-binding sites can sequester miRNA in a non-independent manner and hence increase the prospects of a ceRNA effect.In this study, we examine the impact that miRNA levels have on the DRT and thereby address a key difference between the hierarchical affinity and mixed-affinity models. We then analyze the influence of target-site complementarity on ceRNA-mediated gene regulation and examine the extent to which closely spaced miRNA-binding sites can cooperatively influence the potency of target-gene derepression. Finally, we develop a mathematical model, which incorporates both the mixed-affinity binding and the repressive activities of miRNAs to recapitulate our results.