Barrett Steven P, Parker Kevin R, Horn Caroline, Mata Miguel, Salzman Julia
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, United States of America.
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America.
PLoS Genet. 2017 Dec 13;13(12):e1007114. doi: 10.1371/journal.pgen.1007114. eCollection 2017 Dec.
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.
ciRS-7是一种被深入研究、高表达且保守的环状RNA。关于其生物合成，包括其启动子的位置，基本上一无所知。一个普遍的假设是，ciRS-7是一种特殊的环状RNA，因为它是从一个缺乏任何同意义成熟线性RNA转录本的基因座转录而来的。为了研究ciRS-7的生物合成，我们开发了一种算法来定义其启动子，并预测人类ciRS-7启动子与长链非编码RNA LINC00632的启动子一致。我们使用多种正交实验分析验证了这一预测。我们还使用计算方法和实验验证来确定ciRS-7外显子序列嵌入在人类和小鼠中两侧都有隐蔽外显子的线性转录本中。总之，这些实验和计算证据为该基因座的调控产生了一个新模型：（a）ciRS-7与其他环状RNA一样，因为它被剪接成线性转录本；（b）ciRS-7的表达主要由LINC00632启动子的染色质状态决定；（c）对于ciRS-7生物合成足够的转录和剪接因子在缺乏可检测到的ciRS-7表达的细胞中表达。这些发现对ciRS-7的调控和功能研究具有重要意义，我们开发的用于联合分析RNA-seq和ChIP-seq数据的分析框架揭示了在当前参考注释中遗漏的全基因组范围内发现重要生物调控的潜力。
