The lupus autoantigen La is an Xist-binding protein involved in Xist 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, the lupus autoantigen La (encoding gene Ssb) 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 ES 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. 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, Xist transcripts are unstable and misfolded. These results show that La is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.