UC San Diego

Translation sits at the interface between nucleic acids and proteins and is recognized to regulate gene expression. The regulation of mRNA translation is prone to errors that reduce the levels of functional proteins invoking diseases. Techniques that allow for monitoring protein synthesis will help us understand translation regulation in health and disease. Ribosome profiling is the standard method for the in-vivo measurement of cellular translation but contains many limitations that make it inefficient. APEX-seq is a proximity labeling technique that utilizes an APEX2 fusion protein to localize and biotinylate RNA in-vivo for their downstream enrichment and sequencing. During translation elongation, mRNA and tRNA moieties are moved through the ribosome by the translocation process, catalyzed by the GTP binding protein, eEF2. In addressing the limitations of ribosome profiling, we are developing an in-vivo technique called Protein Synthesis Profiling, that uses an APEX2-eEF2 fusion protein to label actively translated mRNAs during ribosomal translocation for their in-vitro biotinylation, downstream enrichment, and sequencing. Saccharomyces cerevisiae was used as a model organism and was transformed with an APEX2-eEF2 DNA construct via homologous recombination, resulting in the expression of the APEX2-eEF2 fusion protein in-vivo. An evaluation was conducted on the feasibility of Protein Synthesis Profiling, and the data supports the functional performance of the APEX2-eEF2 fusion protein in labeling RNAs near the ribosome and the in-vitro biotinylation of the labeled RNAs, allowing for their subsequent enrichment and sequencing.