Analyzing editosome function in high-throughput

Abstract

Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.

Hammerhead ribozyme-based U-insertion and deletion RNA editing assays for multiplexing in HTS applications

“…Recently Del Campo et al introduced a novel fluorescence-based insertion/deletion editing assay that utilizes capillary electrophoresis to provide quantitative information on editing products and intermediates (Del Campo et al 2020). While the appealing nature of this assay is its ability to monitor not just the final edited product but also several editing intermediates in a high-throughput manner, high chemical substrate concentrations (0.5 mM ATP and 0.1 mM UTP) could potentially thwart the chances of finding competitive inhibitors of the catalytic enzymes.…”