环状RNA (circRNA) 是通过反向剪接产生的共价闭合单链RNA。一些 circRNA 被认为具有功能；然而，我们对 circRNA 调控的途径缺乏了解。在这里，我们生成了一个短发夹 RNA 文库，针对 3,354 个人类 circRNA 的后剪接点，这些 circRNA 在人类中以不同水平（从低到高）表达。我们使用该库对来自四种组织类型的 18 个癌细胞系进行功能丧失性增殖筛选，这些癌细胞系含有导致定义途径的组成性活性的突变。上下文特异性和非特异性 circRNA 均已被识别。一些 circRNA 被发现直接调节其前体，而另一些则具有与其前体无关的功能。我们通过二次筛选验证了这些观察结果，并发现了 circRERE(4-10) 和 circHUWE1(22,23)（两种细胞必需的 circRNA）、circSMAD2(2-6)（一种 WNT 通路调节因子）和 circMTO1(2， RI,3)，MAPK 信号传导的调节剂。我们的工作揭示了 circRNA 调控的通路，并提供了具有可测量功能的 circRNA 目录。© 2024。作者，获得 Springer Nature Limited 的独家许可。

Circular RNA (circRNA) is covalently closed, single-stranded RNA produced by back-splicing. A few circRNAs have been implicated as functional; however, we lack understanding of pathways that are regulated by circRNAs. Here we generated a pooled short-hairpin RNA library targeting the back-splice junction of 3,354 human circRNAs that are expressed at different levels (ranging from low to high) in humans. We used this library for loss-of-function proliferation screens in a panel of 18 cancer cell lines from four tissue types harbouring mutations leading to constitutive activity of defined pathways. Both context-specific and non-specific circRNAs were identified. Some circRNAs were found to directly regulate their precursor, whereas some have a function unrelated to their precursor. We validated these observations with a secondary screen and uncovered a role for circRERE(4-10) and circHUWE1(22,23), two cell-essential circRNAs, circSMAD2(2-6), a WNT pathway regulator, and circMTO1(2,RI,3), a regulator of MAPK signalling. Our work sheds light on pathways regulated by circRNAs and provides a catalogue of circRNAs with a measurable function.© 2024. The Author(s), under exclusive licence to Springer Nature Limited.