ciRS-7 exonic sequence is embedded in a long non-coding RNA locus

Abstract

22ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is 23 known about its biogenesis, including the location of its promoter. A prevailing assumption has 24 been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any 25 mature linear RNA transcripts of the same sense. Our interest in the biogenesis of ciRS-7 led 26 us to develop an algorithm to define its promoter. This approach predicted that the human ciRS-27 7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this 28 prediction using multiple orthogonal experimental assays. We also used computational 29 approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded 30 in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this 31 experimental and computational evidence generate a new view of regulation in this locus: (a) 32 ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is 33 primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and 34 splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-35 7 expression. These findings have significant implications for the study of the regulation and 36 function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and 37