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 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.
Highlights
Messenger RNAs encode information in both their primary sequence and their higher order structure
For comparison with a previous study documenting the effects of modified nucleotides on RNA immunogenicity [16], we made eGFP Messenger RNAs (mRNAs) wherein both U and C were substituted with Ψ and 5-methyl-cytidine (m5C), respectively
We analyzed the impact of primary coding sequence (CDS) sequence on protein expression of mRNAs containing no modified nucleotides
Summary
Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. We combined computational sequence design with global modified nucleotide substitution as tools to investigate the separate impacts of mRNA primary sequence and structural stability on protein output. We find that differences in the innate thermodynamic base pair stability of 2 modified uridine nucleotides, N1methyl-pseudouridine and 5-methoxy-uridine, induce global changes in mRNA secondary structure. These structural changes in turn drive changes in protein expression. 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].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
