School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
Institute of Translational Medicine, Tianjin Union Medical Center, State Key Laboratory of Medicinal Chemical Biology, Collaborative Innovation Center for Biotherapy, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Protein Sciences, National Demonstration Center for Experimental Biology Education and College of Life Sciences, Nankai University, Tianjin 300071, China.
Nucleic Acids Res. 2021 Nov 18;49(20):11596-11613. doi: 10.1093/nar/gkab1003.
Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse 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 mouse 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 plays critical roles in XCI. In Ssb knockdown 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.
利用 Pumilio 蛋白的可编程 RNA 序列结合结构域，我们在活的小鼠细胞中 FLAG 标记了 Xist（失活的 X 染色体特异性转录本）。亲和下拉结合质谱分析鉴定了 138 种候选 Xist 结合蛋白，其中 Ssb（也称为狼疮自身抗原 La）被验证为对 X 染色体失活（XCI）具有功能重要性的蛋白质。在 Ssb 敲低细胞中检测到广泛的 XCI 缺陷，包括染色质紧缩、体外分化过程中雌性小鼠胚胎干细胞死亡以及全染色体单等位基因表达模式。活细胞成像显示 Xist RNA 存在定义性的 XCI 缺陷：Xist 云的形成。Ssb 是一种普遍存在且多功能的 RNA 结合蛋白，具有 RNA 伴侣和 RNA 解旋酶活性。Ssb 的功能分析表明，RNA 伴侣结构域在 XCI 中发挥关键作用。在 Ssb 敲低细胞中，Xist 转录本不稳定且错误折叠。这些结果表明 Ssb 参与 XCI 至关重要，可能作为一种调节 Xist 细胞内结构的蛋白质。
