Biomedical Sciences

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Identification of Long Noncoding RNA Recognized by RNA-Binding Protein TLS/FUS: Purification of RNAs by Affinity Chromatography of GST-TLS

Received: Nov. 01, 2022    Accepted: Nov. 25, 2022    Published: Dec. 15, 2022
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Abstract

RNA binding protein TLS/FUS binds promoter-associated noncoding RNA-D (pncRNA-D), long noncoding RNA (lncRNA), and inhibits histone acetyltransferase (HAT) activity in cellular nuclei to repress the cyclin-D1 gene transcription. TLS is expressed in neuronal cells and plays pivotal roles in neuronal develop and function. Its precipitation in motor neurons is supposed to cause amyotrophic lateral sclerosis (ALS) one of neurodegenerative diseases. Our preliminary experiments indicate that pncRNA-D should repress the phase separation and the resultant precipitation of TLS in biochemical conditions. This implies that pncRNA-D be a seed for effective drug against ALS. It is conceivable that a pool of lncRNAs bound to TLS should be a competent library screening for an ALS drug. Then, we decided to search for more lncRNAs to regulate phase separation and precipitation. In this manuscript, we develop a simple and swift technology of the affinity purification of unidentified RNAs from HeLa cell total RNA using bacterially expressed glutathione S-transferase-tagged-TLS (GST-TLS). Screening of the GST-TLS bound RNA has been performed with a human lncRNA microarray using a fluorescent dye, the cy3-labeled bound RNA as a probe. 1728 lncRNAs of more than two-fold increase at the fluorescent signals have been identified, compared to those of the input HeLa cell total RNA. The top 25 lncRNAs from the 1728 lncRNAs were expressed at more than 12-fold induction. Tentatively, the top four putative lncRNAs were employed for further analysis. Then, these lncRNAs have been shown to have specific binding to GST-TLS and also cellular TLS. The precipitation based experiment to detect phase separation has shown that these lncRNAs inhibit the phase separation-induced precipitation which is dissolved in 1, 6-hexanediol (1,6-HD). There is no significant sequence homology over these lncRNAs, although a consensus conformation of these lncRNAs is the loops and stems structure based upon the predicted secondary structures of the top 25 lncRNAs selected. These data confirm that the GST-TLS based affinity purification of RNA bound to TLS works well to provide the novel lncRNAs specific to TLS. The method to identify novel lncRNAs recognized by TLS provides a profitable technique for initiating the biology of TLS-bound lncRNAs in cellular programs.

DOI 10.11648/j.bs.20220804.15
Published in Biomedical Sciences ( Volume 8, Issue 4, December 2022 )
Page(s) 144-156
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

TLS/FUS, pncRNA-D, GST-TLS, Long Noncoding RNA, Phase Separation, Intrinsically Disordered Region, IDP

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    Naomi Ueda, Ryoma Yoneda, Riki Kurokawa. (2022). Identification of Long Noncoding RNA Recognized by RNA-Binding Protein TLS/FUS: Purification of RNAs by Affinity Chromatography of GST-TLS. Biomedical Sciences, 8(4), 144-156. https://doi.org/10.11648/j.bs.20220804.15

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    ACS Style

    Naomi Ueda; Ryoma Yoneda; Riki Kurokawa. Identification of Long Noncoding RNA Recognized by RNA-Binding Protein TLS/FUS: Purification of RNAs by Affinity Chromatography of GST-TLS. Biomed. Sci. 2022, 8(4), 144-156. doi: 10.11648/j.bs.20220804.15

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    AMA Style

    Naomi Ueda, Ryoma Yoneda, Riki Kurokawa. Identification of Long Noncoding RNA Recognized by RNA-Binding Protein TLS/FUS: Purification of RNAs by Affinity Chromatography of GST-TLS. Biomed Sci. 2022;8(4):144-156. doi: 10.11648/j.bs.20220804.15

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  • @article{10.11648/j.bs.20220804.15,
      author = {Naomi Ueda and Ryoma Yoneda and Riki Kurokawa},
      title = {Identification of Long Noncoding RNA Recognized by RNA-Binding Protein TLS/FUS: Purification of RNAs by Affinity Chromatography of GST-TLS},
      journal = {Biomedical Sciences},
      volume = {8},
      number = {4},
      pages = {144-156},
      doi = {10.11648/j.bs.20220804.15},
      url = {https://doi.org/10.11648/j.bs.20220804.15},
      eprint = {https://download.sciencepg.com/pdf/10.11648.j.bs.20220804.15},
      abstract = {RNA binding protein TLS/FUS binds promoter-associated noncoding RNA-D (pncRNA-D), long noncoding RNA (lncRNA), and inhibits histone acetyltransferase (HAT) activity in cellular nuclei to repress the cyclin-D1 gene transcription. TLS is expressed in neuronal cells and plays pivotal roles in neuronal develop and function. Its precipitation in motor neurons is supposed to cause amyotrophic lateral sclerosis (ALS) one of neurodegenerative diseases. Our preliminary experiments indicate that pncRNA-D should repress the phase separation and the resultant precipitation of TLS in biochemical conditions. This implies that pncRNA-D be a seed for effective drug against ALS. It is conceivable that a pool of lncRNAs bound to TLS should be a competent library screening for an ALS drug. Then, we decided to search for more lncRNAs to regulate phase separation and precipitation. In this manuscript, we develop a simple and swift technology of the affinity purification of unidentified RNAs from HeLa cell total RNA using bacterially expressed glutathione S-transferase-tagged-TLS (GST-TLS). Screening of the GST-TLS bound RNA has been performed with a human lncRNA microarray using a fluorescent dye, the cy3-labeled bound RNA as a probe. 1728 lncRNAs of more than two-fold increase at the fluorescent signals have been identified, compared to those of the input HeLa cell total RNA. The top 25 lncRNAs from the 1728 lncRNAs were expressed at more than 12-fold induction. Tentatively, the top four putative lncRNAs were employed for further analysis. Then, these lncRNAs have been shown to have specific binding to GST-TLS and also cellular TLS. The precipitation based experiment to detect phase separation has shown that these lncRNAs inhibit the phase separation-induced precipitation which is dissolved in 1, 6-hexanediol (1,6-HD). There is no significant sequence homology over these lncRNAs, although a consensus conformation of these lncRNAs is the loops and stems structure based upon the predicted secondary structures of the top 25 lncRNAs selected. These data confirm that the GST-TLS based affinity purification of RNA bound to TLS works well to provide the novel lncRNAs specific to TLS. The method to identify novel lncRNAs recognized by TLS provides a profitable technique for initiating the biology of TLS-bound lncRNAs in cellular programs.},
     year = {2022}
    }
    

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  • TY  - JOUR
    T1  - Identification of Long Noncoding RNA Recognized by RNA-Binding Protein TLS/FUS: Purification of RNAs by Affinity Chromatography of GST-TLS
    AU  - Naomi Ueda
    AU  - Ryoma Yoneda
    AU  - Riki Kurokawa
    Y1  - 2022/12/15
    PY  - 2022
    N1  - https://doi.org/10.11648/j.bs.20220804.15
    DO  - 10.11648/j.bs.20220804.15
    T2  - Biomedical Sciences
    JF  - Biomedical Sciences
    JO  - Biomedical Sciences
    SP  - 144
    EP  - 156
    PB  - Science Publishing Group
    SN  - 2575-3932
    UR  - https://doi.org/10.11648/j.bs.20220804.15
    AB  - RNA binding protein TLS/FUS binds promoter-associated noncoding RNA-D (pncRNA-D), long noncoding RNA (lncRNA), and inhibits histone acetyltransferase (HAT) activity in cellular nuclei to repress the cyclin-D1 gene transcription. TLS is expressed in neuronal cells and plays pivotal roles in neuronal develop and function. Its precipitation in motor neurons is supposed to cause amyotrophic lateral sclerosis (ALS) one of neurodegenerative diseases. Our preliminary experiments indicate that pncRNA-D should repress the phase separation and the resultant precipitation of TLS in biochemical conditions. This implies that pncRNA-D be a seed for effective drug against ALS. It is conceivable that a pool of lncRNAs bound to TLS should be a competent library screening for an ALS drug. Then, we decided to search for more lncRNAs to regulate phase separation and precipitation. In this manuscript, we develop a simple and swift technology of the affinity purification of unidentified RNAs from HeLa cell total RNA using bacterially expressed glutathione S-transferase-tagged-TLS (GST-TLS). Screening of the GST-TLS bound RNA has been performed with a human lncRNA microarray using a fluorescent dye, the cy3-labeled bound RNA as a probe. 1728 lncRNAs of more than two-fold increase at the fluorescent signals have been identified, compared to those of the input HeLa cell total RNA. The top 25 lncRNAs from the 1728 lncRNAs were expressed at more than 12-fold induction. Tentatively, the top four putative lncRNAs were employed for further analysis. Then, these lncRNAs have been shown to have specific binding to GST-TLS and also cellular TLS. The precipitation based experiment to detect phase separation has shown that these lncRNAs inhibit the phase separation-induced precipitation which is dissolved in 1, 6-hexanediol (1,6-HD). There is no significant sequence homology over these lncRNAs, although a consensus conformation of these lncRNAs is the loops and stems structure based upon the predicted secondary structures of the top 25 lncRNAs selected. These data confirm that the GST-TLS based affinity purification of RNA bound to TLS works well to provide the novel lncRNAs specific to TLS. The method to identify novel lncRNAs recognized by TLS provides a profitable technique for initiating the biology of TLS-bound lncRNAs in cellular programs.
    VL  - 8
    IS  - 4
    ER  - 

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Author Information
  • Division of Biomedical Sciences, School of Medicine, Saitama Medical University, Saitama, JapanDivision of Biomedical Sciences, School of Medicine, Saitama Medical University, Saitama, Japan

  • Division of Biomedical Sciences, School of Medicine, Saitama Medical University, Saitama, Japan

  • Division of Biomedical Sciences, School of Medicine, Saitama Medical University, Saitama, Japan

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