Engineering 2’O-mRNA methyltransferases for industrial biocatalysis
September 24-28, 2017
Eukaryotic messenger RNA (mRNA) are universally modified at their 5’ end into a cap 0 structure consisting of an N7-methylguanosine and an inverted 5’-5’ triphosphate bridge linking the penultimate nucleoside. Multicellular eukaryotes possess the capacity to further modify this cap by 2’O-methylating the ribose of the penultimate nucleotide producing a so-called cap 1 structure1. This methylation seems to be a molecular signature for the discrimination between self and non-self mRNA2. In order to escape the innate immune system of the infected cell, some viruses have also evolved the ability to methylate their cap structures1.
By analogy, therapeutic mRNAs must be non-immunogenic in order to restore or supplement the function of altered genes by mRNA-based therapy3. In this context, we propose to exploit the capacity of Vaccinia virus to produce non-immunogenic mRNAs. More specifically, VP39 is a 39 kDa-enzyme directly involved in the mRNAs’ post-transcriptional modifications. It catalyses the 2’O-methylation in the 5’ cap structure producing the cap 1 mRNA and acts by heterodimerisation as a processivity factor with the poly(A) RNA polymerase4. However, the low expression level of VP39 in Escherichia coli (E. coli) as well as its low in vitro catalytic efficiency have so far limited its use for industrial biocatalysis.
Here, the two above-mentioned limitations are tackled by complementary approaches: i) we use a Split-GFP5 strategy coupled with ultrahigh throughput screening to select for higher soluble expression in E. coli and ii) we design smart libraries seeking to directly improve the catalytic turnover of the enzyme.
1. Leung, D. W. & Amarasinghe, G. K. When your cap matters: structural insights into self vs non-self recognition of 5’ RNA by immunomodulatory host proteins. Curr. Opin. Struct. Biol. 36, 133–141 (2016).
2. Zust, R. et al. Ribose 2’-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat Immunol 12, 137–143 (2011).
3. Sahin, U., Kariko, K. & Tureci, O. mRNA-based therapeutics - developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014).
4. Hodel, A. E., Gershon, P. D., Shi, X. & Quiocho, F. A. The 1.85 A structure of vaccinia protein VP39: A bifunctional enzyme that participates in the modification of both mRNA ends. Cell 85, 247–256 (1996).
5. Cabantous, S. & Waldo, G. S. In vivo and in vitro protein solubility assays using split GFP. Nat. Methods 3, 845–854 (2006).
Pierre-Yves Colin, Paul A. Dalby, Mary Stahley, and Jared Davis, "Engineering 2’O-mRNA methyltransferases for industrial biocatalysis" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/enzyme_xxiv/24