Identification of host tRNAs preferentially recognized by the Plasmodium surface protein tRip

We used MIST (Microarray Identification of Shifted tRNAs), a previously established in vitro approach, to systematically assess the specificity of complexes between native H. sapiens tRNAs and recombinant P. falciparum tRip. We demonstrate that tRip unexpectedly binds to host tRNAs with a wide range of specificities, suggesting that only a small subset of human tRNAs are preferentially imported into the parasite. We employed a proven technique where tRip-bound tRNAs isolated from total human tRNA extracts through EMSA were hybridized on tRNA microarrays for systematic identification. This approach named MIST for Microarray Identification of Shifted tRNA was first used to identify non-cognate tRNAs complexed to yeast RRS (Eriani et al., 2015). Since the tRNA binding domain of tRip is located on the parasite surface away from the cytoplasmic content, its binding specificity was tested exclusively against total tRNAs of its human host in absence of any other parasite molecules. To establish the proper experimental conditions for MIST we reasoned that: (i) tRNAs should be in excess to favor competition and allow tRip to naturally select the best interactants from crude human tRNAs (ii) one tRNA molecule is the strict minimum required to saturate one tRip in vitro. Accordingly, different tRNA:tRip ratios were used. While the protein concentration remained constant (2 µM dimer), the amount of tRNA was set to 8, 20, and 40 µM, which corresponds to 4, 10, and 20 times more tRNAs than tRip dimers. In all three conditions tested, both tRNAs present in the complex and the free fraction were recovered from the gel and analyzed with tRNA microarrays. As control experiments, two untreated samples of crude tRNAs purified from HeLa cells were also analyzed, generating a total of 8 different microarrays. Please note that the 'raw_data.xlsx' contains both sample raw data and raw data processor spreadsheet. The raw data processor shows the connection between the raw and the processed data and also contains instructions on how to use the processor for any users who wish to regenerate the processed data from raw data.