The lupus autoantigen La is anXist-binding protein involved inXistfolding and cloud formation

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-taggedXist(<underline>i</underline>nactivated<underline>X</underline>chromosome<underline>s</underline>pecific transcript) in live cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidateXist-binding proteins, from which, the lupus autoantigen La (encoding geneSsb) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected inSsbknockdown cells, including chromatin compaction, death of female ES cells duringin vitrodifferentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging ofXistRNA reveals the defining XCI defect:Xistcloud formation. La is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of La shows that the RNA chaperone domain and/or the ATP binding motif play critical roles in XCI. In mutant cells,Xisttranscripts are unstable and misfolded. These results show that La is critically involved in XCI, possibly as a protein regulating the in-cell structure ofXist.