Decoding mRNA translatability and stability from 5'UTR

Precise control of protein synthesis by engineering sequence elements in 5 untranslated region (5UTR) remains a fundamental challenge. To accelerate our understanding of cis-regulatory code embedded in 5UTR, we devised massively parallel reporter assays from a synthetic mRNA library composed of over one million 5UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP leads to a broad range of mRNA translatability and 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 in 5UTR with G-quadruplex as a mark for mRNA decay in the P-body. Unexpectedly, an unstructured A-rich element in 5UTR, while enabling cap-independent translation, destabilizes mRNAs in the absence of translation. Our results not only expose diverse sequence features of 5UTR in controlling mRNA translatability, but also reveal ribosome-dependent and -independent mRNA surveillance pathways.