The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global
structural architecture, cell-induced conformational changes, and protein–RNA interactions within Xist are poorly understood. We used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP)
to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure,
via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing elements adopt
accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction
domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable
interactions. This work creates a broad quantitative framework for understanding structure–function interrelationships for
Xist and other lncRNAs in cells.