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Related Concept Videos

RNA Editing02:23

RNA Editing

9.4K
RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes
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Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE).

Wolf-Matthias Leeder1, Elisabeth Kruse1, H Ulrich Göringer1

  • 1Molecular Genetics, Technical University Darmstadt, Darmstadt, Germany.

Bio-Protocol
|April 2, 2021
PubMed
Summary

Researchers developed a new in vitro assay to study RNA editing in protozoa. This method uses fluorescent molecules to track uridine insertion and deletion, aiding in understanding the editosome and finding new drugs.

Keywords:
African trypanosomesEditosomeGuide RNAMitochondrial gene expressionProtozoan parasitesRNA-editingRNA-processingU-insertion/U-deletion editing

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A Nonsequencing Approach for the Rapid Detection of RNA Editing
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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Parasitology

Background:

  • Gene expression in African trypanosomes relies on mitochondrial RNA editing.
  • Uridine nucleotides are inserted/deleted into transcripts to create mRNAs.
  • The editosome, a large complex, catalyzes this RNA editing process.

Purpose of the Study:

  • To develop an improved in vitro assay for quantifying editosome catalytic activity.
  • To enable detailed mechanistic and enzymological investigations of RNA editing.
  • To facilitate high-throughput screening for RNA editing inhibitors.

Main Methods:

  • Utilized synthetic, fluorophore-labeled oligoribonucleotides as substrates.
  • Employed capillary electrophoresis coupled with laser-induced fluorescence (CE/LIF) for product separation and detection.
  • Incorporated RNase-resistant substrate RNAs and multiplex fluorophore labeling for simultaneous U-insertion and U-deletion monitoring.

Main Results:

  • Developed a robust, quantitative in vitro assay for editosome activity.
  • The assay requires only nanogram quantities of material.
  • Demonstrated high-throughput capability using multicapillary CE/LIF instruments.

Conclusions:

  • The new assay provides a powerful tool for studying RNA editing mechanisms.
  • The assay is suitable for investigating the enzymology of the editosome.
  • This method can be used to screen for novel RNA editing-specific inhibitors.