The Impact of miRNA Target Sites in Coding Sequences and in 3′UTRs

Animal miRNAs are a large class of small regulatory RNAs that are known to directly and negatively regulate the expression of a large fraction of all protein encoding genes. The identification and characterization of miRNA targets is thus a fundamental problem in biology. miRNAs regulate target genes by binding to 3' untranslated regions (3'UTRs) of target mRNAs, and multiple binding sites for the same miRNA in 3'UTRs can strongly enhance the degree of regulation. Recent experiments have demonstrated that a large fraction of miRNA binding sites reside in coding sequences. Overall, miRNA binding sites in coding regions were shown to mediate smaller regulation than 3'UTR binding. However, possible interactions between target sites in coding sequences and 3'UTRs have not been studied. Using transcriptomics and proteomics data of ten miRNA mis-expression experiments as well as transcriptome-wide experimentally identified miRNA target sites, we found that mRNA and protein expression of genes containing target sites both in coding regions and 3'UTRs were in general mildly but significantly more regulated than those containing target sites in 3'UTRs only. These effects were stronger for conserved target sites of length 7-8 nt in coding regions compared to non-conserved sites. Combined with our other finding that miRNA target sites in coding regions are under negative selection, our results shed light on the functional importance of miRNA targeting in coding regions.