Other mRNA sequence features that reportedly correlate with protein output are dinucleotide frequency in the CDS ( 5) and the effect of codon order on locally accessible charged tRNA pools ( 6). Conversely, “nonoptimal” codons can increase ribosomal pausing and decrease mRNA half-life ( 3, 4). Synonymous codon choice directly impacts translation, with highly expressed genes tending to include more “optimal” codons ( 1, 2). Both translational efficiency and functional half-life are driven by features encoded in the primary mRNA sequence. How much protein is produced per mRNA molecule is a function of how well the translational machinery initiates and elongates on the coding sequence (CDS) and the mRNA’s functional half-life. Messenger RNAs (mRNAs) direct cytoplasmic protein expression. This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability. 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). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). 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. 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. Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure.

Half life dark interval