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

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By Research Archive Posted on Jan 3, 2023
In Category - Research
Naomi Ueda, Ryoma Yoneda, Riki Kurokawa Biomedical Sciences (Science Publishing Group) 2022
English Japan
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.
TLS/FUS, pncRNA-D, GST-TLS, Long Noncoding RNA, Phase Separation, Intrinsically Disordered Region, IDP

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.

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