Although previous studies demonstrated circular RNAs (circRNAs) does not exclusively comprise mRNA exons, no study has extensively explored their internal structure. By combining an algorithm with long-read sequencing data and experimental validation, we, for the first time, comprehensively investigate internal components of circRNAs in 10 human cell lines and 62 fruit fly samples, and reveal the prevalence of alternative splicing (AS) events within circRNAs. Significantly, a large proportion of circRNA AS exons can hardly be detected in mRNAs and are enriched with binding sites of distinct splicing factors from those enriched in mRNA exons. We find that AS events in circRNAs have a preference towards nucleus localization and exhibit tissue- and developmental stage-specific expression patterns. This study suggests an independent regulation on the biogenesis or decay of AS events in circRNAs and the identified circular AS isoforms provide targets for future studies on circRNA formation and function.

中文翻译：

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全面鉴定环状RNA中的内部结构和其他剪接事件。

尽管以前的研究表明环状RNA（circRNA）并不仅仅包含mRNA外显子，但没有研究广泛探索其内部结构。通过将算法与长期测序数据和实验验证相结合，我们首次全面研究了10个人类细胞系和62个果蝇样品中circRNA的内部组成，并揭示了内部选择性剪接（AS）事件的普遍性circRNA。值得注意的是，几乎没有在mRNA中检测到很大比例的circRNA AS外显子，并且富含与那些富含mRNA外显子的剪接因子不同的结合位点。我们发现，circRNAs中的AS事件具有对细胞核定位的偏好，并表现出组织和发育阶段的特异性表达模式。