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

Steven P Barrett,Miguel Mata,Kevin R Parker,Caroline Horn,Julia Salzman,Gregory P Copenhaver

doi:10.1371/journal.pgen.1007114

Abstract

ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. To study the biogenesis of ciRS-7, we developed an algorithm to define its promoter and predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generates a new model for regulation of this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChIP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.

Highlights

Until recently, the expression of circRNA was almost completely uncharacterized, with a few important exceptions [1,2,3,4]
As a first step to identify the ciRS-7 promoter, we examined available chromatin immunoprecipitation sequencing (ChIP-seq) data from HeLa, HEK293, and in vitro differentiated neuronal cells, which exhibit a range of ciRS-7 expression, from very low in HeLa cells to very high in neurons [7,8,9]
CiRS-7 is hosted in a lncRNA locus data linking ciRS-7 to cryptic up- and downstream exons; and (c) continuous RNA-seq signal across the locus starting from the upstream gene C230004F18Rik, through ciRS-7, and into the downstream transcript C030023E24Rik

Summary

Introduction

The expression of circRNA was almost completely uncharacterized, with a few important exceptions [1,2,3,4]. CiRS-7, one of the most highly expressed and most intensely studied circRNAs, is an exception to this rule, where recent work has shown it functions as a miRNA sponge [6,7]. CiRS-7 exhibits increasing expression in neuronal differentiation models in vitro [9]. A recent ciRS-7 knock-out mouse exhibited neuronal defects, including impaired sensorimotor gating and dysfunctional synaptic transmission [10]. In spite of these functional findings, a model for the biogenesis and regulation of ciRS-7 is lacking, and key questions remain: What is the primary transcript that is processed to produce ciRS-7? In spite of these functional findings, a model for the biogenesis and regulation of ciRS-7 is lacking, and key questions remain: What is the primary transcript that is processed to produce ciRS-7? Where in the genome is the promoter for this transcript? Are there any other spliced transcripts, circular or linear, generated from this locus?

Methods

Results

Discussion

Conclusion