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Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1
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 by light, which is an excellent tool to exert spatio-temporal contro...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179545/ https://www.ncbi.nlm.nih.gov/pubmed/34163701 http://dx.doi.org/10.1039/d1sc00159k |
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author | Reichert, Dennis Mootz, Henning D. Rentmeister, Andrea |
author_facet | Reichert, Dennis Mootz, Henning D. Rentmeister, Andrea |
author_sort | Reichert, Dennis |
collection | PubMed |
description | 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 by light, which is an excellent tool to exert spatio-temporal control. However, light-mediated control of eukaryotic mRNA processing and the respective enzymes has not been reported. We used genetic code expansion to install a photo-caged tyrosine (Y) in the active site of the cap methyltransferase Ecm1. This enzyme is responsible for guanine N7 methylation of the 5′ cap, which is required for translation. Substituting Y284 with the photocaged ortho-nitrobenzyl-tyrosine (ONBY) almost completely abrogated the methylation activity of Ecm1. Irradiation with light removed the ONB group, restoring the native tyrosine and Ecm1 activity, yielding up to 97% conversion of the minimal substrate GpppA within 60 min after activation. Using luciferase- and eGFP-mRNAs as reporters, we could show that light actuates translation by inducing activation of Ecm1 ONBY284 in a eukaryotic in vitro translation system. |
format | Online Article Text |
id | pubmed-8179545 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | The Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
spelling | pubmed-81795452021-06-22 Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1 Reichert, Dennis Mootz, Henning D. Rentmeister, Andrea Chem Sci Chemistry 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 by light, which is an excellent tool to exert spatio-temporal control. However, light-mediated control of eukaryotic mRNA processing and the respective enzymes has not been reported. We used genetic code expansion to install a photo-caged tyrosine (Y) in the active site of the cap methyltransferase Ecm1. This enzyme is responsible for guanine N7 methylation of the 5′ cap, which is required for translation. Substituting Y284 with the photocaged ortho-nitrobenzyl-tyrosine (ONBY) almost completely abrogated the methylation activity of Ecm1. Irradiation with light removed the ONB group, restoring the native tyrosine and Ecm1 activity, yielding up to 97% conversion of the minimal substrate GpppA within 60 min after activation. Using luciferase- and eGFP-mRNAs as reporters, we could show that light actuates translation by inducing activation of Ecm1 ONBY284 in a eukaryotic in vitro translation system. The Royal Society of Chemistry 2021-02-08 /pmc/articles/PMC8179545/ /pubmed/34163701 http://dx.doi.org/10.1039/d1sc00159k Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/ |
spellingShingle | Chemistry Reichert, Dennis Mootz, Henning D. Rentmeister, Andrea Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1 |
title | Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1 |
title_full | Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1 |
title_fullStr | Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1 |
title_full_unstemmed | Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1 |
title_short | Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1 |
title_sort | light-control of cap methylation and mrna translation via genetic code expansion of ecm1 |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179545/ https://www.ncbi.nlm.nih.gov/pubmed/34163701 http://dx.doi.org/10.1039/d1sc00159k |
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