Issue 5, 2021

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

Abstract

The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled the manipulation and investigation of many steps in the involved processes. However, examples for light-mediated control of eukaryotic mRNA processing and the responsible enzymes are still rare. In particular, methylation of the 5′ cap of mRNA is required for ribosome assembly, and the responsible guanine-N7 methyltransferase (MTase) from E. cuniculi (Ecm1) proved suitable for activating translation. Here, we report on a photoswitchable MTase obtained by bridging the substrate-binding cleft of Ecm1 with a tetra-ortho-methoxy-azobenzene. This azobenzene derivative is characterized by efficient trans-to-cis isomerization using red light at 615 nm. Starting from a cysteine-free Ecm1 variant (ΔCys), we used a computational approach to identify suitable conjugation sites for the azobenzene moiety. We created and characterized the four best-ranked variants, each featuring two appropriately positioned cysteines close to the substrate-binding cleft. Conjugating and crosslinking the azobenzene between C149/C155 in a designed Ecm1 variant (VAR3-Az) enabled light-dependent modulation of the MTase activity and showed a 50% higher activity for the cis form than the trans-form of the azobenzene conjugated to VAR3-Az.

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

Supplementary files

Article information

Article type
Paper
Submitted
14 5月 2021
Accepted
25 6月 2021
First published
29 6月 2021
This article is Open Access
Creative Commons BY-NC license

RSC Chem. Biol., 2021,2, 1484-1490

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

D. Reichert, H. Schepers, J. Simke, H. Lechner, W. Dörner, B. Höcker, B. J. Ravoo and A. Rentmeister, RSC Chem. Biol., 2021, 2, 1484 DOI: 10.1039/D1CB00109D

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