RNA-binding protein-guided 3′ UTR engineering to enhance mRNA stability

Abstract

Extending the lifetime of mRNA is critical for improving the efficacy of mRNA therapeutics. The 3′ untranslated region (3′ UTR) plays a central role in regulating RNA stability and translation efficiency. Although current SARS-CoV-2 mRNA vaccines use mtRNR1 and AES-derived 3′ UTRs for enhanced stability, the molecular mechanisms remain unclear. Here, we develop an RNA-centric workflow combining RNA pull-down assays with LC-MS/MS proteomics to identify RNA-binding proteins (RBPs) that can bind to stable 3′ UTR sequences. Proteomic analysis reveals 29 RBPs significantly enriched on stable 3′ UTR sequences relative to the control. We further select nine cytoplasmic RBPs for functional RNAi screening and rationally design 3′ UTR sequences utilizing the RNA binding motifs of these RBPs. This integrated approach, combining proteomics, functional validation, and rational sequence engineering, establishes a robust platform to accelerate the development of next-generation mRNA therapeutics.