Mapping the RNA Chaperone Activity of the Trypanosoma brucei Editosome Using SHAPE-Chemical Probing

Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D- and 3D-structures. The RNA molecules are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction, which is catalyzed by a 0.8 MDa protein complex known as the editosome. RNA binding to the editosome is followed by a chaperone-mediated RNA remodeling reaction. The reaction increases the dynamic of specifically U-nucleotides to lower their base-pairing probability and, consequently, generates a simplified RNA folding landscape critical for the progression of the editing reaction cycle. Here, we describe a chemical mapping method to quantitatively monitor the chaperone-driven structural changes of pre-edited mRNAs upon editosome binding. The technique has been termed Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constrained (double-stranded) versus structurally unconstrained (single-stranded) nucleotides. Electrophilic anhydrides such as 1-methyl-7-nitroisatoic anhydride are used as probing reagents, and the ribose 2'-modified nucleotides are mapped as abortive cDNA-synthesis products. As a result, SHAPE allows the identification of all single-stranded and base-paired regions in a given RNA, and the data are used to compute experimentally derived RNA 2D-structures. A side-by-side comparison of the RNA 2D-folds in the prechaperone and postchaperone states finally maps the chaperone-induced dynamics of the different pre-mRNAs with single-nucleotide resolution.