biostudies-literatureLewis CJTAmerican Cancer SocietyNational Institute of Diabetes and Digestive and Kidney DiseasesNIDDK NIH HHSPfizerNational Cancer InstituteNCI NIH HHSNational Institutes of HealthNational Institute of General Medical SciencesNIGMS NIH HHSNational Science Foundation445-459.e5https://www.ebi.ac.uk/biostudies/studies/S-EPMC11780321biostudies-literatureUnknown85(2)mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions (UTRs) including alternative isoforms and identify sequences that mediate 200-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-Molecular cellQuantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs.PMC11780321F31 DK129022DGE2139841R01 GM132358PF-23-1144579-01-RMCITEN2021.RNA01R01 GM101316R01GM1323582330451F31CA254339F31 CA254339F31DK129022R35 GM152167R35GM152167Thoreen CCAbdallah KGilbert WVJin DXie LHDraycott ASChen YLewis CJTBhandarkar SMfalseQuantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs.mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions (UTRs) including alternative isoforms and identify sequences that mediate 200-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-2025-01-01T00:00:00Z2025 Jan2026-06-06T15:23:43.474Z2026-06-01T03:11:06.846ZS-EPMC117803213970618710.1016/j.molcel.2024.11.030