Structured elements drive extensive circular RNA translation

The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs requires internal ribosome entry sites (IRES) if they are to undergo translation without 5’ cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify over 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue -specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is down regulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease. High-throughput oligo-split-eGFP-circRNA reporter screening assay for obtaining cap-independent translation measurements of 55,000 fully designed sequences in parallel on circRNA using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq).

人类基因组编码了数万种功能大多未知的环形RNA（circRNAs）。若要在不依赖5’帽结构的情况下进行翻译，环形RNA需要内部核糖体进入位点（IRES）。本研究开发了一种高通量筛选方法，可在人类细胞中系统性发掘能够指导环形RNA翻译的RNA序列。我们共鉴定出超过17000条内源性与人工合成序列作为候选环形RNA IRES。18S核糖体RNA（18S rRNA）互补序列以及定位于IRES的结构化RNA元件，对于驱动环形RNA翻译具有重要作用。核糖体谱分析与肽组学分析结果显示，存在广泛的IRES-核糖体结合现象，数百种环形RNA编码的蛋白呈现组织特异性分布，且可参与抗原呈递。我们发现，circFGFR1编码的蛋白circFGFR1p在癌症中表达下调，其可作为成纤维细胞生长因子受体1（FGFR1）癌蛋白的负调控因子，在应激过程中抑制细胞生长。系统性鉴定环形RNA IRES元件，可为环形RNA调控、生物学功能与疾病之间建立重要关联。本研究采用高通量寡核苷酸拆分-eGFP-环形RNA报告基因筛选实验，通过荧光激活细胞分选与高通量DNA测序（FACS-seq），可同时对55000条完全设计的序列开展环形RNA非帽依赖翻译水平的检测。