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Optochemical control of cell fate by activation of mRNA translation

Periodic Reporting for period 2 - RNActivate (Optochemical control of cell fate by activation of mRNA translation)

Reporting period: 2019-12-01 to 2021-05-31

Light is an excellent external regulatory element that can be applied to cells and organisms with high spatio-temporal precision and without interfering with cellular processes. Optochemical biology exploits small photo-responsive chemical groups to cage and activate or to switch biomolecular functions in response to light of a defined wavelength. Caged antisense agents have enabled down-regulation of gene expression with spatio-temporal control at the messenger-RNA (mRNA) level in vivo, however approaches for triggering translation of exogenous mRNA lack efficient turn-on effects. To explore the effects of conditional and transient ectopic gene expression in a developing organism it is vital to fully abrogate and restore translational efficiency.
The goal of this project is to bring eukaryotic mRNA under the control of light to trigger efficient ectopic translation with spatio-temporal resolution in cells and in vivo. In addition to labeling and tracking subpopulations of cells, we will use our technology to control and to manipulate cell fate by locally producing proteins responsible for cell death, genome engineering, and cell migration. We will use cultured cells and one-cell stage zebrafish embryos that can be easily injected with mRNA to study the function of ectopic gene expression in early development. Our approach will overcome current limitations of photo-inducible mRNA translation and enable us to manipulate a developing organism at the molecular level.
We developed a new set of AdoMet analogs that can be used to enzymatically transfer photo-caging groups to the target sites of methyltransferases. These include the ortho-nitrobenzylgroup as well as red-shifted derivatives. We could show that several methyltransferases are sufficiently promiscous to accept the cosubstrate analogs and transfer photo-caging groups to nucleic acids. In particular, we could modify the N7 position of the guanosine of the 5' cap of mRNA (GpppG) using the methyltransferase Ecm1. The modification strongly interfered with cap-binding proteins. Upon irradiation with light, we found that the ortho-nitrobenzyl group and its derivatives could not be removed from the N7 position of guanosine. Instead, an unanticipated product was formed under release of CO and CO2. We discovered that the benzophenone-moiety, which is commonly used as photo-crosslinker, serves as a photo-caging group if installed at the N7 position of guanosine and can be completely removed by irradation with light of 365 nm. We also showed that the cap remains functional after photo-deprotection and can be remethylated enzymatically, restoring the original cap function. RNAs with cap remained intact under these rradiation conditions. These major findings were published in Angewandte Chemie ( L. Anhäuser, N. Klöcker, F. Muttach, F. Mäsing, P. Špaček, A. Studer and A. Rentmeister, Angewandte Chemie International Edition 2020, 59, 3161-3165).

Furthermore, we could photocage the N6-position of adenosine at internal positions of adenosine in mRNA using the methyltransferase METTL3-METTL14. In this case, irradiation with light led to the anticipated removal of the photo-caging group. We showed that the photocaging group impaired reverse transcription and that this impairment was released after irradiation and subsequent uncaging (A. Ovcharenko, F. P. Weissenboeck and A. Rentmeister, Angewandte Chemie International Edition 2021, in press).

The photocaging of the N6-position of adenosine also worked with DNA. Here, we could show that the DNA can be protected from enzymatic restriction at specific sequence-elements. Upon irradiation, the photocaging groups were efficiently removed and the restriction of the DNA was restored. Importantly, we could develop a strategy to enzymatically make the AdoMet analogs with photocaging groups. To achieve this, we engineered methionine adenosyl transferases (MATs) from different organisms to accept bulkier side-chains. By solving the crystal structure of the best MAT variant in complex with the AdoMet analog, we could elucidate the molecular details for accepting the ortho-nitrobenzyl group. These results are the basis for metabolic labeling, because they allow us to now start from methionine analogs which are stable and cell-permeable, while the AdoMet analogs have limited stability and are not cell permeable. These results were recently published in Angewandte Chemie and featured in a press release and on the cover page (F. Michailidou, N. Klöcker, N. V. Cornelissen, R. K. Singh, A. Peters, A. Ovcharenko, D. Kümmel and A. Rentmeister, Angewandte Chemie International Edition 2021, 60, 480-485).
We significanlty expanded the toolbox and possibilities of AdoMet analogs. AdoMet analogs were originally developed for site-specific labeling and modification. Our work added benzylic AdoMet analogs to the toolbox and specifically developed AdoMet analogs with photo-caging groups. We could show that several methyltransferases are able to accept these AdoMet analogs and block their target sites in a reversible manner. Photocaging groups have been introduced before by chemical means, but it was hard to achieve site- and sequence-specific modification of long nucleic acids.

Within this project, we could already show that the photocaging groups block interaction of proteins and enzymes with mRNA. For the 5' cap we performed binding studies with the translation initiation factor and a decapping enzyme. For internal modifications of mRNA we showed that reverse transcription is impaired. For DNA, we showed that restriction sites can be blocked. Based on our work, all of this is now possible in a reversible manner - using light as trigger. In nature, methylation of DNA and RNA can be reverted enzymatically. This happens in the fundamental processes of epigenetics and epitranscriptomics. With our methodoology, light can now be used an orthogonal trigger to remove modifications at natural modification sites of methyltransferases.
Finally, we discovered a new type of photocaging group for the N7 position of guanosine. This position showed unexpected photochemistry with ortho-nitrobenzylgroups but a benzophenone-moiety could be removed by light.

Until the end of the project, we will further explore the ability to control functions of mRNA by light and control biological processes. In particular, we will focus on biological processes in living mammalian cells and in developing zebrafish.
Enzymatic photocagin of the mRNA 5' cap