Analyzing editosome function in high-throughput

Nucleic Acids Res. 2020 Sep 25;48(17):e99. doi: 10.1093/nar/gkaa658.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescein / chemistry
  • Fluorescent Dyes / chemistry
  • Genome, Mitochondrial
  • High-Throughput Screening Assays / methods*
  • Multiplex Polymerase Chain Reaction / methods
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • RNA Editing*
  • RNA, Guide / chemistry
  • RNA, Guide / genetics
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Trypanosoma brucei brucei / genetics*
  • Uridine Triphosphate / chemistry

Substances

  • Fluorescent Dyes
  • Protozoan Proteins
  • RNA, Guide
  • RNA, Messenger
  • Fluorescein
  • Uridine Triphosphate