Decoding mRNA translatability and stability from 5' UTR

Precise control of protein synthesis by engineering sequence elements in 5' untranslated region (5'UTR) remains a fundamental challenge. To accelerate our understanding of cis-regulatory code embedded in 5'UTR, we devised massively parallel reporter assays from a synthetic mRNA 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 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 5'UTR with G-quadruplex as a mark for mRNA decay in the P-body. Unexpectedly, an unstructured A-rich element in 5'UTR, while enabling cap-independent translation, destabilizes mRNAs in the absence of translation. Our results not only expose diverse sequence features of 5'UTR in controlling mRNA translatability, but also reveal ribosome-dependent and -independent mRNA surveillance pathways. Overall design: To accelerate our understanding of cis-regulatory code embedded in 5'UTR of mRNA, we devised a high-throughput uORF reporter assay from a synthetic mRNA library composed of over one million 5'UTR variants. This reporter assay is compatible with fluorescence-activated cell sorting and polysome profiling.