Leeder, W.-Matthias ; Göringer, H. Ulrich (2020)
Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing.
In: Methods in molecular biology (Clifton, N.J.), 2106
doi: 10.1007/978-1-0716-0231-7_10
Artikel, Bibliographie
Kurzbeschreibung (Abstract)
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 as a consequence generates a simplified RNA folding landscape that is 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 method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (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 pre- and post-chaperone states finally maps the chaperone-induced dynamic of the different pre-mRNAs with single-nucleotide resolution.
Typ des Eintrags: | Artikel |
---|---|
Erschienen: | 2020 |
Autor(en): | Leeder, W.-Matthias ; Göringer, H. Ulrich |
Art des Eintrags: | Bibliographie |
Titel: | Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing |
Sprache: | Englisch |
Publikationsjahr: | Januar 2020 |
Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Methods in molecular biology (Clifton, N.J.) |
Jahrgang/Volume einer Zeitschrift: | 2106 |
DOI: | 10.1007/978-1-0716-0231-7_10 |
Kurzbeschreibung (Abstract): | 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 as a consequence generates a simplified RNA folding landscape that is 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 method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (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 pre- and post-chaperone states finally maps the chaperone-induced dynamic of the different pre-mRNAs with single-nucleotide resolution. |
ID-Nummer: | pmid:31889257 |
Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Genregulation und RNA-Therapeutika |
Hinterlegungsdatum: | 06 Jan 2020 11:57 |
Letzte Änderung: | 02 Dez 2021 14:22 |
PPN: | |
Export: | |
Suche nach Titel in: | TUfind oder in Google |
Frage zum Eintrag |
Optionen (nur für Redakteure)
Redaktionelle Details anzeigen |