Partial base pairing between 5' and 3' UTRs artificially regulates IVT mRNA expression levels

Eukaryotic mature mRNAs possess a 5' cap and a 3' polyadenylate tail (poly(A)), both of which are essential for regulating mRNA expression levels. This regulation is structurally based on the circularization of mRNA, mediated by physical interactions among the 5' cap-binding protein eIF4E, the 3' poly(A)-binding protein PABPC1, and the translation initiation factor eIF4G, which bridges eIF4E and PABPC1. Here, we demonstrate that partial base pairing between the 5' and 3' untranslated regions (UTRs) of in vitro-transcribed (IVT) mRNAs promotes circularization and artificially boosts the expression levels. The expression levels of IVT mRNAs are influenced by the patterns formed by the lengths of the complementary and non-complementary regions between the UTRs. Moreover, we observed a sigmoidal relationship between the expression levels and Gibbs free energy changes resulting from 5'-3' UTR pairing. Collectively, these findings provide a novel framework for the rational design of 5' and 3' UTRs in IVT mRNAs and for predicting their expression levels based on thermodynamic parameters.