MicroRNA target sites are often conserved during evolution and purifying selection to maintain such sites is expected. On the other hand, comparative analyses identified a paucity of microRNA target sites in coexpressed transcripts, and novel target sites can potentially be deleterious. We proposed that selection against novel target sites pervasive. The analysis of derived allele frequencies revealed that, when the derived allele is a target site, the proportion of nontarget sites is higher than expected, particularly for highly expressed microRNAs. Thus, new alleles generating novel microRNA target sites can be deleterious and selected against. When we analyzed ancestral target sites, the derived (nontarget) allele frequency does not show statistical support for microRNA target allele conservation. We investigated the joint effects of microRNA conservation and expression and found that selection against microRNA target sites depends mostly on the expression level of the microRNA. We identified microRNA target sites with relatively high levels of population differentiation. However, when we analyze separately target sites in which the target allele is ancestral to the population, the proportion of single-nucleotide polymorphisms with high F st significantly increases. These findings support that population differentiation is more likely in target sites that are lost than in the gain of new target sites. Our results indicate that selection against novel microRNA target sites is prevalent and, although individual sites may have a weak selective pressure, the overall effect across untranslated regions is not negligible and should be accounted when studying the evolution of genomic sequences.
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