MicroRNAs (miRNAs) are 20–24 nt long endogenous non-coding RNAs that act as post-transcriptional regulators in metazoa and plants. Plant miRNA targets typically contain a single sequence motif with near-perfect complementarity to the miRNA. Here, we extended and applied the program RNAhybrid to identify novel miRNA targets in the complete annotated Arabidopsis thaliana transcriptome. RNAhybrid predicts the energetically most favorable miRNA:mRNA hybrids that are consistent with user-defined structural constraints. These were: (i) perfect base pairing of the duplex from nucleotide 8 to 12 counting from the 5′-end of the miRNA; (ii) loops with a maximum length of one nucleotide in either strand; (iii) bulges with no more than one nucleotide in size; and (iv) unpaired end overhangs not longer than two nucleotides. G:U base pairs are not treated as mismatches, but contribute less favorable to the overall free energy. The resulting hybrids were filtered according to their minimum free energy, resulting in an overall prediction of more than 600 novel miRNA targets. The specificity and signal-to-noise ratio of the prediction was assessed with either randomized miRNAs or randomized target sequences as negative controls. Our results are in line with recent observations that the majority of miRNA targets are not transcription factors.

cis-Regulatory DNA elements contain multiple binding sites for activators and repressors of transcription. Among these elements are enhancers, which establish gene expression states, and Polycomb/Trithorax response elements (PREs), which take over from enhancers and maintain transcription states of several hundred developmentally important genes. PREs are essential to the correct identities of both stem cells and differentiated cells. Evolutionary differences in cis-regulatory elements are a rich source of phenotypic diversity, and functional binding sites within regulatory elements turn over rapidly in evolution. However, more radical evolutionary changes that go beyond motif turnover have been difficult to assess. We used a combination of genome-wide bioinformatic prediction and experimental validation at specific loci, to evaluate PRE evolution across four Drosophila species. Our results show that PRE evolution is extraordinarily dynamic. First, we show that the numbers of PREs differ dramatically between species. Second, we demonstrate that functional binding sites within PREs at conserved positions turn over rapidly in evolution, as has been observed for enhancer elements. Finally, although it is theoretically possible that new elements can arise out of nonfunctional sequence, evidence that they do so is lacking. We show here that functional PREs are found at nonorthologous sites in conserved gene loci. By demonstrating that PRE evolution is not limited to the adaptation of preexisting elements, these findings document a novel dimension of cis-regulatory evolution.