The mammalian mRNA 5′ cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5′-mediated mRNA turnover. Hence, installation of the proper 5′ cap is crucial in therapeutic applications of synthetic mRNA. The core 5′ cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5′ triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity towards RNA containing secondary structures, and broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.
The ribosome relies on hydrogen bonding interactions between mRNA codons and incoming aminoacyl-tRNAs to ensure rapid and accurate protein production. The inclusion of chemically modified bases into mRNAs has the potential to alter the strength and pattern of hydrogen bonding between mRNAs and aminoacyl-tRNAs to alter protein synthesis. We investigated how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the ability of codons to react with cognate and near-cognate tRNAs and release factors. We find that the presence of a single m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, insertion of m1Ψ can affect the selection of near-cognate tRNAs both in vitro and in human cells. Our observations demonstrate that m1Ψ, and the related naturally occurring pseudouridine (Ψ) modification, exhibit the ability to both increase and decrease the extent of amino acid misincorporation in a codon-position and tRNA dependent manner. To ascertain the chemical logic for our biochemical and cellular observations, we computationally modeled tRNAIle(GAU) bound to unmodified and m1Ψ- or Ψ-modified phenylalanine codons (UUU). Our modeling suggests that changes in the energetics of mRNA:tRNA interactions largely correlate with the context specificity of Ile-miscoding events we observe on Ψ and m1Ψ containing Phe codons. These studies reveal that the sequence context of a given modification within an mRNA plays a large role in determining how (and if) the modification impacts the number and distribution of proteoforms synthesized by the ribosome.
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