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

Cole J T Lewis; Li H Xie; Shivani Milind Bhandarkar; Danni Jin; Kyrillos Abdallah; Austin S Draycott; Yixuan Chen; Carson C Thoreen; Wendy V Gilbert

doi:10.1016/j.molcel.2024.11.030

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

Mol Cell. 2024 Dec 12:S1097-2765(24)00955-9. doi: 10.1016/j.molcel.2024.11.030. Online ahead of print.

Authors

Cole J T Lewis¹, Li H Xie², Shivani Milind Bhandarkar¹, Danni Jin¹, Kyrillos Abdallah¹, Austin S Draycott¹, Yixuan Chen¹, Carson C Thoreen³, Wendy V Gilbert⁴

Affiliations

¹ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA.
² Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA.
³ Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address: carson.thoreen@yale.edu.
⁴ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA. Electronic address: wendy.gilbert@yale.edu.

PMID: 39706187
DOI: 10.1016/j.molcel.2024.11.030

Abstract

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-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissecting mechanisms of human translation initiation and engineering more potent therapeutic mRNAs.

Keywords: 5′ untranslated region; N1-methylpseudouridine; RNA modification; high-throughput screening; ribosome; therapeutic mRNA; translation initiation.