mRNA structure regulates protein expression through changes in functional half-life

Abstract

Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used two approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly-expressed mRNAs contained a highly-structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide-variety of primary sequences. Using a set of eGFP mRNAs that independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e. mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability. [150 words] HighlightsO_LIProtein expression from modified mRNAs tends to follow the pattern m1 {Psi} > U >mo5U C_LIO_LIProtein expression correlates with mRNA thermodynamic stability: {Psi}{approx} m1{Psi} > U > mo5U C_LIO_LIA highly structured CDS correlates with high expression C_LIO_LIIncreased structured mRNAs extend functional half-life C_LI