Precise control of protein synthesis by engineering sequence elements in 5′ untranslated regions (5′ UTRs) remains a fundamental challenge. To accelerate our understanding of the cis-regulatory code embedded in 5′ UTRs, we devised massively parallel reporter assays from a synthetic messenger RNA library composed of over one million 5′ UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP drives a broad range of translational outputs and mRNA stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements with G-quadruplexes as marks for mRNA decay in P-bodies. Unexpectedly, an unstructured A-rich element in 5′ UTRs destabilizes mRNAs in the absence of translation, although it enables cap-independent translation. Our results not only identify diverse sequence features of 5′ UTRs that control mRNA translatability, but they also reveal ribosome-dependent and ribosome-independent mRNA-surveillance pathways.

通过在5'非翻译区（5'UTR）中工程序列元件来精确控制蛋白质合成仍然是一项基本挑战。增进我们对顺式的了解-嵌入5'UTR中的调控代码，我们从合成信使RNA库中设计了大规模平行的报告基因检测，该文库由超过一百万个5'UTR变体组成。上游开放阅读框（uORF）和下游GFP之前的完全随机的10个核苷酸序列驱动哺乳动物细胞中广泛的翻译输出和mRNA稳定性。有效的翻译可以保护mRNA免受降解，而uORF翻译则以依赖UPF1的方式触发mRNA的降解。我们还确定了G-四链体的翻译抑制元件作为P体中mRNA衰减的标记。出乎意料的是，5'UTR中无结构的富含A的元件在不存在翻译的情况下会破坏mRNA的稳定性，尽管它能够进行不依赖帽的翻译。我们的结果不仅确定了可控制mRNA翻译性的5'UTR的各种序列特征，