Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1

Abstract

Gene expression is tightly regulated in all domains of life, with post-transcriptional regulation being more pronounced in higher eukaryotes. Optochemical and optogenetic approaches enable the actuation of many underlying processes...

Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase

“…All four variants retained high activity, yielding more than 70% conversion after 24 h. Ecm1 VAR3 showed the highest MTase activity, yielding 93% conversion after 3 h. Still, compared to the wild-type enzyme, the activity was reduced, as previously 91% conversion of 1a were observed after 2 h at 37 °C. 15 Nevertheless, these data indicate that our combination of rational and computational design provided active Ecm1 variants suitable to attempt azobenzene conjugation. Overall, we could show that the design strategy yielded functional enzyme variants, with cysteine residues accessible for modification.…”

“…[10][11][12][13][14] Furthermore, we could show that via site-specific incorporation of a photo-caged tyrosine in the active site of a guanine-N7 MTase from E. cuniculi (Ecm1), the final step of 5 0 cap formation, provides a way to bring translation under the control of light. 15 Nevertheless, this approach requires amber stop codon suppression and high amounts of the photocaged amino acid, which has to be added to the E. coli culture. Also, once the photo-protecting group has been cleaved, there is no way to regulate enzymatic activity.…”

Quantification of mRNA cap-modifications by means of LC-QqQ-MS

“…Here, chemically modified caps have been reported, improving stability and resistance against decapping enzymes or altering translation [ 17 , 25 ]. In enzyme-based approaches, we recently reported a method for light-activated N 7-methylation of GpppG-mRNA using photocaged Ecm1 as a tool for temporal control of translation [ 19 ]. CAPAM-catalyzed modifications of Am at its N 6 -position did alter immunogenicity and translation of transcripts [ 20 ].…”

“…The enzymes Ecm1, CAPAM and MTAN were produced and purified as previously described [ 19 , 20 , 34 ].…”

“…We have recently developed MTase-based strategies for the site-specific, enzymatic modification of the 5′ -cap in biologically relevant mRNAs. Introduction of a photocleavable group into the methyltransferase Ecm1 enabled light-controlled terminal N 7-guanosine methylation of GpppG-capped RNAs and thus provided a means to trigger translation at the time point of choice ( N 7-methylation by Ecm1 shown in Scheme 1A ) [ 19 ]. We also investigated CAPAM’s cosubstrate promiscuity and found that N 6 -propargylation of 2′ - O -methyl adenosine at the TSN ( Scheme 1B ) altered the immunogenicity of the transcript while translation remained high [ 20 ].…”