Efficient and Selective Encoding of Phosphoserine in Mammalian Cells Utilizing an Evolved Translational System.

• Applicant: Dr. Martin Spinck
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2021 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 493404643

Protein phosphorylation is crucial in coordinating myriad cellular processes. Identifying the contribution of individual phosphorylation to the overall signalling response remains challenging. The simplest method is to mimic phosphorylation by aspartate, however many key phosphoproteins are unresponsive to mimic mutations. In these cases, genetically encoded phosphoserine is an attractive alternative. Beranek et al. (2018) showed that bacterial derived SepRSv1.0/tRNAv1.0CUA enables encoding of phosphorylation in mammalian cells, albeit substantially limited in its utility by glutamine read-through and low translation efficiency.Here, I propose a different approach to develop an adaptive and selective system for the encoding of phosphoserine (or its analogue) in mammalian cells: I want to evolve critical components of the SepRSv1.0/tRNAv1.0CUA translational system in yeast. Herein I plan to engineer the eEF1-alpha-Sep/tRNAv1.0CUA complex active site and its eukaryotic specific interface to improve phosphoserine binding and ribosomal translation fidelity, respectively. Candidates with improved phosphoserine encoding will be selected by yeast display of phosphorylated peptides from the constructed mutant libraries. In addition, the best eEF1-alpha-Sep variants will be target to the mRNA using programmable RNA binding proteins to further improve encoding efficiency while minimize off-target effects.The system(s) will be tested in mammalian cells using an established assay for Mek1 kinase activation and I will demonstrate its functionality on the example of the aspartate unresponsive sites S133 of CREB. The improved system should simplify the systematic investigation of any phosphorylation site in absence of kinase signalling.

• DFG Programme: WBP Fellowship
• International Connection: United Kingdom
• Host: Professor Dr. Jason Chin