Cotranscriptional folding of the lncRNA Xist A-repeats indicates a modular structure

• Published in: bioRxiv
• Main Authors: Jones, Alisha N, Gabel, Frank, Bohn, Stefan, Wolfe, Gregory, Sattler, Michael
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 27.07.2022
• Subjects: Conformation; Molecular modelling; Non-coding RNA; RNA-binding protein; Uracil; X-chromosome inactivation
• DOI: 10.1101/2022.07.26.501616

The A-repeat region of the lncRNA Xist is required for X-chromosome inactivation in placental mammals and comprises 8.5 copies of a conserved 24-nucleotide motif separated by uracil-rich linkers. Several experimentally-supported secondary structures have been proposed for the A-repeats. Here, we consider the effects of cotranscriptional folding and present a comprehensive biochemical and structural analysis of the Xist A-repeats. We show that under native-like conditions the A-repeat region folds into two independent subdomains. The repeats are modularly arranged, forming stable AUCG tetraloop hairpins and inter-repeat dimers. The similarity of chemical probing profiles in cells and in vitro argues that this structure represents the predominant native conformation of the Xist A-repeat in vivo. However, the A-repeat conformation is dynamic and affected by buffer conditions, rationalizing the disparity between previously proposed structures. Our results provide a starting point for studying the interaction with RNA binding proteins and molecular mechanisms linked to X-inactivation. Our integrative approach establishes a pipeline for investigating cotranscriptional pathways of large RNAs and their structures. Competing Interest Statement The authors have declared no competing interest.