The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation

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 (a...

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Main Authors: Ha, Norbert, Ding, Nan, Hong, Ru, Liu, Rubing, Roca, Xavier, Luo, Yingyuan, Duan, Xiaowei, Wang, Xiao, Ni, Peiling, Wu, Haiyang, Zhang, Li-Feng, Chen, Lingyi
Other Authors: School of Biological Sciences
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Language:English
Published: 2022
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Online Access:https://hdl.handle.net/10356/161273
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spelling sg-ntu-dr.10356-1612732023-02-28T17:13:48Z The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation Ha, Norbert Ding, Nan Hong, Ru Liu, Rubing Roca, Xavier Luo, Yingyuan Duan, Xiaowei Wang, Xiao Ni, Peiling Wu, Haiyang Zhang, Li-Feng Chen, Lingyi School of Biological Sciences Science::Biological sciences Binding Protein Animal Cell 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. Ministry of Education (MOE) Ministry of Health (MOH) National Research Foundation (NRF) Published version L.C. was supported by the National Key R&D Program of China [2018YFC1313003, 2018YFA0107002]; National Natural Science Foundation of China [31871485]; Natural Science Foundation of Tianjin [18JCJQJC48400]; 111 Project Grant [B08011]; Fundamental Research Funds for the Central Universities; L.-F.Z. was supported by Singapore Ministry of Education Academic Research Fund [MOE2015-T2-1-093]; Singapore National Research Foundation under its Cooperative Basic Research Grant administered by the Singapore Ministry of Health’s National Medical Research Council [NMRC/CBRG/0092/2015]. Funding for open access charge: the National Key R&D Program of China [2018YFA0107002]. 2022-08-23T05:57:15Z 2022-08-23T05:57:15Z 2021 Journal Article Ha, N., Ding, N., Hong, R., Liu, R., Roca, X., Luo, Y., Duan, X., Wang, X., Ni, P., Wu, H., Zhang, L. & Chen, L. (2021). The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation. Nucleic Acids Research, 49(20), 11596-11613. https://dx.doi.org/10.1093/nar/gkab1003 0305-1048 https://hdl.handle.net/10356/161273 10.1093/nar/gkab1003 34723322 2-s2.0-85121264789 20 49 11596 11613 en MOE2015-T2-1-093 NMRC/CBRG/0092/2015 Nucleic Acids Research © The Authors 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Binding Protein
Animal Cell
spellingShingle Science::Biological sciences
Binding Protein
Animal Cell
Ha, Norbert
Ding, Nan
Hong, Ru
Liu, Rubing
Roca, Xavier
Luo, Yingyuan
Duan, Xiaowei
Wang, Xiao
Ni, Peiling
Wu, Haiyang
Zhang, Li-Feng
Chen, Lingyi
The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation
description 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.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Ha, Norbert
Ding, Nan
Hong, Ru
Liu, Rubing
Roca, Xavier
Luo, Yingyuan
Duan, Xiaowei
Wang, Xiao
Ni, Peiling
Wu, Haiyang
Zhang, Li-Feng
Chen, Lingyi
format Article
author Ha, Norbert
Ding, Nan
Hong, Ru
Liu, Rubing
Roca, Xavier
Luo, Yingyuan
Duan, Xiaowei
Wang, Xiao
Ni, Peiling
Wu, Haiyang
Zhang, Li-Feng
Chen, Lingyi
author_sort Ha, Norbert
title The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation
title_short The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation
title_full The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation
title_fullStr The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation
title_full_unstemmed The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation
title_sort lupus autoantigen la/ssb is an xist-binding protein involved in xist folding and cloud formation
publishDate 2022
url https://hdl.handle.net/10356/161273
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