TY - JOUR T1 - The lupus autoantigen La is an <em>Xist</em>-binding RNA chaperone involved in <em>Xist</em> folding and cloud formation JF - bioRxiv DO - 10.1101/2020.03.07.981860 SP - 2020.03.07.981860 AU - Norbert Ha AU - Nan Ding AU - Ru Hong AU - Rubing Liu AU - Xavier Roca AU - Yingyuan Luo AU - Xiaowei Duan AU - Xiao Wang AU - Li-Feng Zhang AU - Lingyi Chen Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/03/08/2020.03.07.981860.abstract N2 - 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 activity. Functional dissection of La shows that the RNA chaperon domain plays critical roles in XCI. In mutant cells, Xist transcripts are misfolded and unstable. These results show that La is involved in XCI as an RNA chaperone for Xist.SIGNIFICANCE STATEMENT Xist RNA functions as a scaffold to recruit and assemble a protein machinery to epigenetically silence genes along one X chromosome in each female mammalian cell. Here, we devised a FLAG-out system to profile the Xist interactome, and identified the lupus autoantigen La as an Xist-binding protein. The RNA chaperone activity of La is essential for X chromosome inactivation (XCI). When La is downregulated, the folding of Xist RNA is altered, leading to shorter half-life of Xist RNA and compromised Xist cloud formation, which in turn results in XCI defects, including chromatin compaction, chromosome-wide monoallelic gene expression pattern, and death of female ES cells during in vitro differentiation. ER -