Abstract

MicroRNAs (miRNAs) negatively regulate the levels of messenger RNA (mRNA) post-transcriptionally. Recent advances in CLIP (cross-linking immunoprecipitation) technology allowed capturing miRNAs with their cognate mRNAs. Consequently, thousands of validated mRNA–miRNA pairs have been revealed. Herein, we present a comprehensive outline for the combinatorial regulation by miRNAs. We implemented combinatorial and statistical constraints in the miRror2.0 algorithm. miRror estimates the likelihood of combinatorial miRNA activity in explaining the observed data. We tested the success of miRror in recovering the correct miRNA from 30 transcriptomic profiles of cells overexpressing a miRNA, and to identify hundreds of genes from miRNA sets, which are observed in CLIP experiments. We show that the success of miRror in recovering the miRNA regulation from overexpression experiments and CLIP data is superior in respect to a dozen leading miRNA-target prediction algorithms. We further described the balance between alternative modes of joint regulation that are executed by pairs of miRNAs. Large-scale data from CLIP technologies were used for testing the validity and quality of our combinatorial-statistical analysis. We provide evidence for the concept of ‘miRNAs working together’ for an individual targeted gene and a large set of genes. The assumption being that a small number of coordinated miRNAs act to maintain robust homeostasis. Under this assumption, even a modest modulation executed in a combinatorial fashion can yield to a substantial change in cell phenotype. Finally, manipulated cells were tested for the possible involvement of miRNA in shaping their transcriptomes. We identified instances in which the observed transcriptome can be explained by a combinatorial regulation of miRNA pairs. We illustrate miRror2.0 platform as a useful application in reducing the prediction noise and in capturing the underlying combinatorial modus operandi of miRNA in a wide range of cellular conditions. We conclude that such a joint operation of miRNAs is an attractive strategy to maintain cell homeostasis and overcoming the low specificity inherent in individual miRNA–mRNA interaction.