Identification and evolutionary comparison of circular RNAs in five mammalian species and three organs.

Circular RNAs (circRNAs) are found across eukaryotes and can function in post-transcriptional gene regulation. Their biogenesis through a circle-forming backsplicing reaction is facilitated by reverse-complementary repetitive sequences promoting pre-mRNA folding. Orthologous genes from which circRNAs arise overall, contain more strongly conserved splice sites and exons than other genes, yet it remains unclear to what extent this conservation reflects purifying selection acting on the circRNAs themselves. Our analyses of circRNA repertoires across five species representing three mammalian lineages (marsupials, eutherians: rodents, primates) reveal that surprisingly few circRNAs arise from orthologous genes from different species. Even the circRNAs from shared loci are associated with young, recently active and species-specific transposable elements, rather than with common, ancient transposon integration events. These observations suggest that many circRNAs emerged convergently during evolution – as a byproduct of splicing in orthologs prone to transposable element insertion. Overall, our findings argue against widespread functional circRNA conservation. Annotation and expression analyses of circRNAs using RNA-sequencing data in five mammalian species (opossum, mouse, rat, rhesus macaque, human) and three organs (liver, cerebellum, testis). Included are three biological replicates for each condition (conditions = species and tissues). All organisms used were male and wild type. Each replicate was splitted into two: the first sample was directly used for library preparation, the second one treated with RNase R prior library preparation to degrade linear RNAs, which subsequently enrichs circRNAs. Samples were sequenced using Illumina paired-end sequencing and circRNAs annotated and analysed for their evolutionary history.