The Lupus Autoantigen La/Ssb is an <i>Xist</i>-Binding Protein Involved in <i>Xist</i> Folding and Cloud Formation

Abstract

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain and/or the ATP binding motif play critical roles in XCI. In mutant cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.