TU Darmstadt / ULB / TUbiblio

Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing

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 Frage zum Eintrag

Optionen (nur für Redakteure)
Redaktionelle Details anzeigen Redaktionelle Details anzeigen