Functional Conservation of Both CDS- and 3′-UTR-Located MicroRNA Binding Sites between Species

MicroRNAs (miRNAs) mediate gene regulation posttranscriptionally through pairing of their seed (2-7 nt) to 3'-untranslated regions (3'-UTRs) or coding regions (coding sequences [CDSs]) of their target genes. CDS target sites generally show weaker repression effects than 3'-UTR sites. However, little is known about the conservation of the function, that is, repression effect, for these two groups of target sites. In addition, no systematic analysis of the evolutionary constraint on CDS sites exists to date. To address these questions, we performed RNA-sequencing to quantify the regulatory effect of miR-15a/miR-16 and miR-92a on their CDS and 3'-UTR targets in human and macaque cells. These miRs were knocked down transiently so the repression effect could be tracked immediately. Although on average CDS targets are less derepressed than 3'-UTR targets in both species, both the 3'-UTR targets and the CDS targets are functionally conserved. The evolutionary analysis of miRNA target sites shows that CDS sites are more conserved than nontarget control, albeit to a lesser extent than 3'-UTR sites. In conclusion, CDS target sites are functional, even though they are subject to less functional constraint than 3'-UTR target sites.