Evolutionary Dynamics of microRNA target sites across vertebrate evolution

Abstract

MicroRNAs (miRNAs) control the abundance of the majority of the vertebrate transcriptome. The recognition sequences, or target sites, for bilaterian miRNAs are found predominantly in the 3′ untranslated regions (3′UTRs) of mRNAs, and are amongst the most highly conserved motifs within 3′UTRs. However, little is known regarding the evolutionary pressures that lead to loss and gain of such target sites. Here, we quantify the selective pressures that act upon miRNA target sites. Notably, selective pressure extends beyond deeply conserved binding sites to those that have undergone recent substitutions. Our approach reveals that even amongst ancient animal miRNAs, which exert the strongest selective pressures on 3′UTR sequences, there are striking differences in patterns of target site evolution between miRNAs. Considering only ancient animal miRNAs, we find three distinct miRNA groups, each exhibiting characteristic rates of target site gain and loss during mammalian evolution. The first group both loses and gains sites rarely. The second group shows selection only against site loss, with site gains occurring at a neutral rate, whereas the third loses and gains sites at neutral or above expected rates. Furthermore, mutations that alter strength of existing target sites are disfavored. Applying our approach to individual transcripts reveals variation in the distribution of selective pressure across the transcriptome and between miRNAs, ranging from strong selection acting on a small subset of targets of some miRNAs, to weak selection on many targets for other miRNAs. miR-20 and miR-30, and many other miRNAs, exhibit broad, deeply conserved targeting, while several other comparably ancient miRNAs show a lack of selective constraint, and a small number, including mir-146, exhibit striking evidence of rapidly evolving target sites. Our approach adds valuable perspective on the evolution of miRNAs and their targets, and can also be applied to characterize other 3′UTR regulatory motifs. AUTHOR SUMMARY Gene regulation typically involves two components; the first is a regulator, and the second is a target that the regulator acts on. Examining the evolution of both components allows us to make inferences regarding how gene regulation originated and how it is changing over time. One gene regulatory system, which is widespread in mammals and other animals, relies upon regulators known as microRNAs (miRNAs), which recognize target sites within mRNAs, leading to post-transcriptional repression of the targeted mRNAs. This system has been studied extensively from the perspective of the evolution of the miRNA regulators. Moreover, a subset of the target sites themselves are known to be deeply conserved. Here, we have systematically examined the rate at which target sites for individual miRNAs are created and destroyed across mammalian evolution. We find that mutations that strengthen or weaken existing target sites are strongly disfavored. For ancient microRNAs, even recently evolved target sites are under strong selective constraint, and as miRNAs age, they tend to initially experience selection only against loss of existing target sites, and later accumulate strong selection against gain of novel target sites.