Periodic Reporting for period 1 - meltRBP (Tracking interactions between RNA and RNA-binding proteins by thermal profiling of the proteome)
Reporting period: 2017-04-01 to 2019-03-31
In 2014, Dr Mikhail Savitski, group leader at EMBL, developed a novel approach, termed thermal proteome profiling (TPP). This methodology is based on the principle that the binding of a ligand to a protein causes a shift in its stability, which can be read out by the proteins’ denaturing properties, using the cellular thermal shift assay (CETSA). In detail, target engagement is measured by exposing the intact cells to a range of different temperatures (37¬¬–65°C, at increments of 3°C). Thereby, a specific temperature induces unfolding of a protein, which leads to the formation of an insoluble pellet after centrifugation. The supernatant now contains all the proteins that are stable at the given temperature, which can be quantified either by Western blotting for specific targets, or on a proteome-wide scale using mass spectrometry. The addition of a drug, as in Savitski et al., (2014), perturbs the system and results in specific protein melting point differences.
The project proposed here made use of this innovative approach, by transferring this cutting-edge technology to the study of RNA-protein complexes and identify all of the RBPs that are bound to a specific RNA. One major advantage of this application is that the differential effects on protein stability modulated by the concentration of a specific RNA molecule can be measured in the context of the whole cell. Specifically, we optimised the technique with a well-characterized model system, the iron regulatory protein/iron response element interaction, followed by the application of TPP to discover all RBPs bound to the iron response element of the HIF2 5’untranslated region. Trying to apply TPP to a novel context, directed our interest to Enolase 1, a glycolytic enzyme that binds RNA.
The second objective involved the application of TPP to a cellular context where the endogenous RNA levels could be manipulated by RNAi or CRISPR Cas9. Unfortunately, the differential expression of full-length mRNAs resulted in changes on many different levels (translation, metabolic state and protein-protein interactions) resulting in TPP uncovering RNA-independent changes. Thus, we decided to revert back to a drug-like treatment of the cells to uncover novel RBP-RNA interactions.
The final project objective was based on recent data that had shown that the eukaryotic RNA-binding proteome is much larger than previously anticipated. It encompasses a number of proteins with well-established functions unrelated to RNA biology, including many metabolic enzymes. To elucidate the physiological role of one such metabolic enzyme in RNA biology, we had focussed on the glycolytic enzyme enolase 1 (ENO1) due to its evolutionarily conserved RNA-binding capacity. As part of the optimisation of TPP for the application to the ENO1-RNA context, we confirmed ENO1 to be a bona fide RNA-binding protein.