Lewis Cole J T, Xie Li H, Bhandarkar Shivani Milind, Jin Danni, Abdallah Kyrillos, Draycott Austin S, Chen Yixuan, Thoreen Carson C, Gilbert Wendy V
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA.
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.
Mol Cell. 2025 Jan 16;85(2):445-459.e5. doi: 10.1016/j.molcel.2024.11.030. Epub 2024 Dec 19.
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.
信使核糖核酸（mRNA）疗法为治疗性蛋白质的高效开发与递送提供了一种潜在的通用策略。当前的mRNA疫苗包含化学修饰的核苷酸，以降低细胞免疫原性。在此，我们开发了一种高效、高通量的方法，用于测量经治疗性修饰的RNA以及内源性RNA上的人类翻译起始。利用系统水平的生物化学方法，我们对核糖体募集到数万个包括可变异构体在内的人类5'非翻译区（UTR）进行定量，并确定介导200倍效应的序列。我们观察到编码序列对翻译起始具有广泛影响，并鉴定出3至6个核苷酸的小调控元件，这些元件足以显著影响翻译输出。掺入N1-甲基假尿苷（m1Ψ）可通过特定的5'UTR选择性增强翻译，我们证明这些UTR优于当前mRNA疫苗的UTR。我们的方法广泛适用于剖析人类翻译起始机制以及设计更有效的治疗性mRNA。
