Abstract
Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”.
Highlights
Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions
We experimentally examined the feasibility of integrating the mathematical model for our single plasmid reporter-system, which is referred to as Untranslated Trans Assay (UTA), by using a synthetic non-human targeting siRNA
One of these served as reporter of transcriptional and translational activity while the other contained the exact complementary sequences of a candidate-miRNA, serving as a reporter of gene regulatory activity. (Fig. 1)
Summary
Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels and by miRNA/target-binding capability. Progress in next-generation sequencing introduced numerous datasets for transcript analyses, which are used for diagnosis and prognostics in various diseases, in particular in several cancer types[11] These analyses intuitively and frequently concluded that miRNA repression activity is reflected by its expression pattern and level, and in turn, that a robustly expressed miRNA shall repress its targets with increased efficacy. Some highly expressed miRNAs reached only weak regulatory activity, while studies on transgene inhibition by miRNAs suggested that a certain level of miRNA expression is required to obtain a detectable repression[20,21]
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