Enhancing mRNA translation efficiency by introducing sequence optimized AU-rich elements in 3′ UTR via HuR anchorage

mRNA technology holds immense promise as an innovative therapeutic approach with applications spanning infectious disease vaccines, cancer immunotherapy, protein replacement, and gene editing. However, practical use of mRNA has been hindered by challenges such as low cellular stability and transient protein expression. For addressing these, we propose a novel strategy to optimize mRNA sequences, particularly in the untranslated region, by inserting adenylate/uridylate-rich elements (AU-rich elements) to enhance stability and protein expression. Our investigation revealed that integrating AU-rich elements between the open reading frame (ORF) and the 3' untranslated region (3' UTR) significantly enhances RNA stability compared with other insertion sites. We identified cytoplasmic Human antigen R (HuR) as an essential RNA-binding protein responsible for promoting mRNA stability and translation, confirmed through HuR knockdown experiments and pull-down assays between AU-rich elements and HuR. Through rational design, we optimized the sequence of natural AU-rich elements and identified the essential "AUUUA" element, which, with certain repeats, can increase protein expression up to 5-fold. To demonstrate the universality of AU-rich element sequences in enhancing mRNA translation, we switched the coding proteins from luciferase to EGFP, mCherry, and ovalbumin (OVA), finding that both natural and engineered AU-rich element sequences amplify the expression of these proteins. In conclusion, leveraging the functionalities of RNA-binding proteins and the natural regulation of RNA stability in the untranslated region represents a novel strategy to enhance mRNA pharmacokinetics in the cytoplasm, expanding the potential applications of mRNA in therapeutic drugs.