Systematic Discovery of Xist RNA Binding Proteins

• Published in: Cell Vol. 161; no. 2; pp. 404 - 416
• Main Authors: Chu, Ci, Zhang, Qiangfeng Cliff, da Rocha, Simão Teixeira, Flynn, Ryan A., Bharadwaj, Maheetha, Calabrese, J. Mauro, Magnuson, Terry, Heard, Edith, Chang, Howard Y.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.04.2015
• Subjects: Animals; chromatin; Chromatin - metabolism; Drosophila; Female; Gene Silencing; histones; Humans; mass spectrometry; Mass Spectrometry - methods; Mice; non-coding RNA; nuclear matrix; Ribonucleoproteins - analysis; RNA, Long Noncoding - metabolism; RNA-binding proteins; RNA-Binding Proteins - analysis; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; X chromosome
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2015.03.025

Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3′ RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. [Display omitted] •ChIRP-MS identifies endogenous protein partners associated with specific RNAs•Xist lncRNA binds 81 proteins in two waves during X chromosome inactivation•HnrnpK is required for Xist-mediated chromatin modifications and Polycomb targeting•Spen binds to Xist A-repeat and is required for gene silencing Development of a general method for identifying RNA-protein interactions in vivo reveals 81 endogenous proteins that associate with Xist RNA in two waves to control mammalian dosage compensation.