Natural antisense transcripts (NATs) are regulatory RNAs that contain sequence complementary to other RNAs, these other RNAs usually being messenger RNAs. In eukaryotic genomes, cis-NATs overlap the gene they complement.
Here, our goal is to analyze the distribution and evolutionary conservation of cis-NATs for a variety of available data sets for Arabidopsis thaliana, to gain insights into cis-NAT functional mechanisms and their significance. Cis-NATs derived from traditional sequencing are largely validated by other data sets, although different cis-NAT data sets have different prevalent cis-NAT topologies with respect to overlapping protein-coding genes. A. thaliana cis-NATs have substantial conservation (28-35% in the three substantive data sets analyzed) of expression in A. lyrata. We examined evolutionary sequence conservation at cis-NAT loci in Arabidopsis thaliana across nine sequenced Brassicaceae species (picked for optimal discernment of purifying selection), focussing on the parts of their sequences not overlapping protein-coding transcripts (dubbed 'NOLPs'). We found significant NOLP sequence conservation for 28-34% NATs across different cis-NAT sets. This NAT NOLP sequence conservation versus A. lyrata is generally significantly correlated with conservation of expression. We discover a significant enrichment of transcription factor binding sites (as evidenced by CHIP-seq data) in NOLPs compared to randomly sampled near-gene NOLP-like DNA , that is linked to significant sequence conservation. Conversely, there is no such evidence for a general significant link between NOLPs and formation of small interfering RNAs (siRNAs), with the substantial majority of unique siRNAs arising from the overlapping portions of the cis-NATs.
In aggregate, our results suggest that many cis-NAT NOLPs function in the regulation of conserved promoter/regulatory elements that they 'over-hang'.