Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA.
Cellular Biology Section, Laboratory of Viral Diseases, NIAID, Bethesda, MD, USA.
Nat Struct Mol Biol. 2020 Sep;27(9):814-821. doi: 10.1038/s41594-020-0465-x. Epub 2020 Jul 27.
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.
通过工程化 5' 非翻译区（5'UTR）中的序列元件来精确控制蛋白质合成仍然是一个基本挑战。为了加速我们对 5'UTR 中内源性调控密码子的理解，我们从由超过一百万个 5'UTR 变体组成的合成信使 RNA 文库中设计了大规模平行报告基因检测。一个完全随机的 10 个核苷酸序列位于上游开放阅读框（uORF）和下游 GFP 之前，可在哺乳动物细胞中驱动广泛的翻译输出和 mRNA 稳定性。虽然有效的翻译可以保护 mRNA 免受降解，但 uORF 的翻译会以 UPF1 依赖的方式触发 mRNA 降解。我们还发现，P 体中具有 G-四联体的翻译抑制元件是 mRNA 降解的标志。出乎意料的是，5'UTR 中无规则的富含 A 的元件在没有翻译的情况下会使 mRNA 不稳定，尽管它可以实现无 cap 依赖性翻译。我们的研究结果不仅确定了控制 mRNA 可译性的 5'UTR 的多种序列特征，而且还揭示了核糖体依赖性和核糖体非依赖性的 mRNA 监测途径。
