Engineering RNA-binding proteins and RNA-modifying enzymes to visualize mRNA localization in living cells
Final Report Abstract
Specific localization of mRNAs to certain subcellular regions represents a mechanism for regulating gene expression with excellent spatial-temporal control and exists in various cell types ranging from bacteria to neurons. Studying mRNA localization, transport, translation, and degradation in subcellular regions will undoubtedly enrich our understanding of how cells and organisms function. To study the dynamics of mRNA distribution and transport, there is an increasing demand for universal probes suitable for visualization of mRNAs in living cells. In this project we followed two strategies to label and detect mRNAs in living cells: We successfully developed a chemo-enzymatic approach to fluorescently label mRNA at different positions of the 5′ cap. The approach was based on methyltransferases, protein engineering, synthetic cosusbrate analogs of S-adenosyl-L-methionine (AdoMet) and different types of click chemistry – including fast bioorthogonal reactions like the tetrazine ligation with strained alkenes. The development and optimization of this toolbox allowed us to produced modified mRNAs and – for the first time reported – label them in living mammalian cells. Most of the modifications at the 5′ cap reduced translation, which stipulated the idea of using cap modifications to control translation by light in future work. In addition to the originally proposed work, we developed mRNA labeling with multiple fluorescent dyes at the poly(A) tail. This labeling strategy gave brighter fluorescent signals and had even positive impact on the translational efficiency of the mRNAs in vitro and in cells. This approach is now followed up for studying mRNA-localization in zebrafish embryos (collaboration with Prof. Raz, Münster). In a second strategy, we engineered the sequence-specific RNA-binding protein Pumilio for different target sequences. We developed a fluorescence complementation system based on a tripartite split-GFP to detect specific RNA in vitro with minimal background. This technique termed tetramolecular fluorescence complementation (TetFC) was further developed to detect two sequences in two different colours and to detect any given target RNA with the help of staple RNAs. We also achieved implementation of TetFC in living E. coli cells. Fluorescence activated cell sorting (FACS) allowed to enrich E. coli cells expressing the target RNA. The system can now be used to engineer Pumilio variants with novel binding specificities. To perform also the enzymatic modification of mRNA in cells, we developed an enzymatic cascade starting from methionine analogs, because AdoMet analogs are not cell permeable. This approach now allows metabolic labeling of RNA. In future work, we wish to address the specificity for certain methyltransferases.
Publications
- A chemo-enzymatic approach for sitespecific modification of the RNA cap. Angew Chem Int Ed (2013) 52:7874-7878
Schulz D, Holstein JM, Rentmeister A
(See online at https://doi.org/10.1002/anie.201302874) - Tetramolecular Fluorescence Complementation for Detection of Specific RNAs in Vitro. ChemBioChem (2013) 14:200-204
Kellermann SJ, Rath AK, Rentmeister A
(See online at https://doi.org/10.1002/cbic.201200734) - Bioorthogonal site-specific labeling of the 5'-cap structure in eukaryotic mRNAs. Chemical Communications (2014), 50:4478-4481
Holstein JM, Schulz D, Rentmeister A
(See online at https://doi.org/10.1039/c4cc01549e) - Enzymatic modification of 5'-capped RNA with a 4-vinylbenzyl group provides a platform for photoclick and inverse electrondemand Diels-Alder reaction. Chemical Science (2015) 6(2):1362-1369
Holstein JM, Stummer D, Rentmeister A
(See online at https://doi.org/10.1039/c4sc03182b) - A Biocatalytic Cascade for Versatile One-Pot Modification of mRNA Starting from Methionine Analogues. Angew Chem Int Ed Engl (2016): 55: 1917-1920
Muttach F, Rentmeister A
(See online at https://doi.org/10.1002/anie.201507577) - A genetically encodable system for sequencespecific detection of RNAs in two colors. ChemBioChem (2016) 17(10):895-899
Kellermann SJ, Rentmeister A
(See online at https://doi.org/10.1002/cbic.201500705) - Modifying the 5′-cap for click reactions of eukaryotic mRNA and to tune translation efficiency in living cells. Angew Chem Int Ed Engl (2016) 55:10899-903
Holstein JM, Anhäuser L, Rentmeister A
(See online at https://doi.org/10.1002/anie.201604107) - A benzylic linker promotes methyltransferase catalyzed norbornene transfer for rapid bioorthogonal tetrazine ligation. Chemical Science (2017) 8(12):7947-7953
Muttach F, Muthmann N, Reichert D, Anhauser L, Rentmeister A
(See online at https://doi.org/10.1039/c7sc03631k) - Dual 5′ cap labeling based on regioselective RNA methyltransferases and bioorthogonal reactions. Chemistry – A European Journal (2017) 23(25):6165-6173
Holstein JM, Muttach F, Schiefelbein SHH, Rentmeister A
(See online at https://doi.org/10.1002/chem.201604816) - Multiple covalent fluorescence labeling of eukaryotic mrRNA at the poly(A) tail enhances translation and can be performed in living cells. Nucleic Acids Research (2019) 47(7):e42-e42
Anhäuser L, Hüwel S, Zobel T, Rentmeister A
(See online at https://doi.org/10.1093/nar/gkz084)
