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

Significance Despite widespread recognition that RNA is inherently structured, the interplay between local and global mRNA secondary structure (particularly in the coding region) and overall protein expression has not been thoroughly explored. Our work uses 2 approaches to disentangle the regulatory roles of mRNA primary sequence and secondary structure: global substitution with modified nucleotides and computational sequence design. By fitting detailed kinetic expression data to mathematical models, we show that secondary structure can increase mRNA half-life independent of codon usage. These findings have significant implications for both translational regulation of endogenous mRNAs and the emerging field of mRNA therapeutics. 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 2 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 with 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.

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