microRNAs (miRNAs) influence the expression of around 60 percent of mammalian genes and have special relevance to the study of development and disease. They function by interacting with messenger (mRNA) via Watson-Crick base pairing, disrupting translation or transcription. A short seed region at the 5’end of a miRNA strand is important in binding to complementary target regions of mRNA. The seed site is evolutionarily conserved and is widely used in bioinformatics studies to predict miRNA target sites in the genome.
miRNAs bind to their target mRNAs within an RNA induced silencing complex (RISC) and bulk sequencing of pools of miRNA-mRNA, isolated from these reaction complexes, allows researchers to infer binding relationships. However, sequencing datasets do not preserve individual molecular relationships and typically, only 50 percent of an mRNA pool can be matched computationally to partner miRNA seed sites. With this in mind, Nicole Cloonan from the University of Queensland, Australia, and colleagues explore the importance of central regions of miRNA (centered sites) that are also highly evolutionarily conserved in their study in Genome Biology.
Venn diagram of the number of microarray mRNA probes that bind canonical miRNAs and isomiRs. Image source: Martin et al, Genome Biology, 2014, 15:R51
To elucidate binding relationships, the authors labeled ten different miRNAs with biotin and introduced each probe into cells to bind to target mRNA. The biotin tagged mRNA was then affinity purified from the cells and hybridized to microarrays containing known human mRNAs. This pull-down approach allows an individual miRNA to be assigned to its population of target mRNAs. In addition to using probes that were known to be true ‘canonical’ miRNAs, the authors also used naturally occurring isomiRs of each mRNA probe (miRNAs generated from the same pre-miRNA as the ‘canonical’ miRNA but with different 5′ and/or 3′ end cleavage) as controls that differed in seed site but share centered sites.
Each miRNA probe revealed a target population of approximately 1500 mRNAs, showing accordance with computational predictions and other published data. Base pair matches between the target mRNAs and individual miRNAs suggest that binding commonly occurs at both seed and centered sites. Assuming that centered site binding still occurs when there is some mismatch between the miRNA and its target, then together, seed and centered sites could account for up to 90 percent of interactions within the isolated mRNA populations. The latter ‘imperfect’ centered binding scenario was verified by Luciferase assay, considered the gold standard for testing miRNA-mRNA interactions.
Further experiments show that the biotin pull-down approach is robust in isolating RISC based miRNA-mRNA, although slightly biased against the sampling of seed mediated interactions, which may more readily dissociate during capture. Crucially, the rate of false positive identification of centered site interactions was found to be low. It was also observed that centered site miRNA binding most often caused translational repression, as opposed to mRNA degradation, in order to facilitate gene silencing.
It is clear that centered site mediated interactions should be a major consideration when interpreting and modeling miRNA regulated networks. However, the inclusion of centered binding sites may not lend predictive power to purely computational approaches to identify miRNA binding sites in the genome, that already suffer imprecision and tend to find too many putative binding sites. Instead, the authors advise that high throughput experimental methods at the level of both gene and protein expression are needed to gain a realistic picture of miRNA-mRNA interactions.