Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs

We report an efficient, high-throughput method to optimize translation initiation from modified mRNAs. Using direct analysis of ribosome targeting, DART, we identify 5′ untranslated sequences that mediate 250-fold effects on ribosome recruitment, show that incorporation of m1Ψ significantly affects activity, and identify more active 5′ UTRs that surpass current mRNA vaccines. DART is broadly applicable to dissect translation initiation mechanisms of human genes and optimize mRNA regulatory sequences for desired protein outputs An RNA pool containing > 35,000 human 5′UTRs were obtained through in vitro transcription of a synthesized DNA oligonucleotide library. The RNA pool was allowed to translate in vitro in HeLa cell extract with the presence of cycloheximide. Initiating Ribosomes were frozen at start codons. 80S-associated RNAs were isolated using sucrose gradient. We analysis the 5′UTRs in the fraction associated with 80S and in the total IVT input. The ratio indicates the initiation efficciency. DMS-MapSeq was used to analysis the secondary structure of RNAs in the solution.