Leeder, Wolf-Matthias ; Kruse, Elisabeth ; Göringer, H. Ulrich (2021)
Trypanosomatid, fluorescence-based U-insertion/U-deletion RNA-editing (FIDE).
In: Bio-protocol, 11 (5)
doi: 10.21769/BioProtoc.3935
Artikel, Bibliographie
Kurzbeschreibung (Abstract)
Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors. .
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2021 |
| Autor(en): | Leeder, Wolf-Matthias ; Kruse, Elisabeth ; Göringer, H. Ulrich |
| Art des Eintrags: | Bibliographie |
| Titel: | Trypanosomatid, fluorescence-based U-insertion/U-deletion RNA-editing (FIDE). |
| Sprache: | Englisch |
| Publikationsjahr: | 5 März 2021 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Bio-protocol |
| Jahrgang/Volume einer Zeitschrift: | 11 |
| (Heft-)Nummer: | 5 |
| DOI: | 10.21769/BioProtoc.3935 |
| Kurzbeschreibung (Abstract): | Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors. . |
| ID-Nummer: | pmid:33796609 |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Genregulation und RNA-Therapeutika |
| Hinterlegungsdatum: | 06 Apr 2021 06:32 |
| Letzte Änderung: | 06 Apr 2021 06:32 |
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