Engineering of modified linear and circular messenger RNA and study of the influence on protein expression tuning
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Abstract
RNA modifications created the epitranscriptome that shaped our understanding of the regulation of biological processes. Modifications were mapped in mRNAs and little is known about their role in modulating their functionality.
Here, selected modifications which are naturally occurring in RNA were inserted in a targeted manner in an enhanced green fluorescence protein (EGFP) mRNA, called point-modified, to assess their influence on protein expression. In analogy to a piece of a puzzle, the mRNA design included sequence breakdown into three fragments, where the middle fragment harbours the modification at the desired site. Subsequently, the full point-modified mRNA was reconstituted by a 3-way-one-pot ligation, and then purified by real-time gel elution. Such a ligation technique, previously applied for shorter size length, provided the possibility for application on much longer mRNA. Essential sites for translation initiation (the Kozak motif and the start codon), which were not yet investigated, as well as internal codons from the coding sequence were modified. The obtained point-modified mRNA was validated by LC-MS/MS and RiboMeth-seq. The presence of 2’-O-ribose-methylated or pseudouridine in point-modified mRNA, revealed a codon-position-dependent effect, stimulating or hampering the overall protein production. Findings obtained here in the context of IRES-driven translation expanded prior work on the influence of modifications on the canonical-translation system.
In a comparative study, the random incorporation of modifications in mRNA was also explored under different conditions, by adding or omitting structural elements (namely IRES, cap and poly(A)). Considering the remarkable structure and differences between circular and linear RNA, enzymatic-based strategy was used for RNA circularization. The obtained product was via RP-HPLC. Protein expression levels of circular RNA relative to the linear RNA form depended on (i) the purification method and (ii) the used translation system. In the last project, randomly modified mRNA was used to outline the limited antibody specificity upon enrichment with immunoprecipitation methods, otherwise required for a significant analysis at the transcriptome-wide level.