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.
| Published in | Biomedical Sciences (Volume 8, Issue 4) |
| DOI | 10.11648/j.bs.20220804.15 |
| 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 |
TLS/FUS, pncRNA-D, GST-TLS, Long Noncoding RNA, Phase Separation, Intrinsically Disordered Region, IDP
| [1] | Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, et al. (2009) Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323, 1208-1211, doi: 323/5918/1208 [pii]10.1126/science.1165942. |
| [2] | Kwiatkowski TJ, Jr., Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T, et al. (2009) Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323, 1205-1208, doi: 323/5918/1205 [pii]10.1126/science.1166066. |
| [3] | Lagier-Tourenne C & Cleveland DW (2009) Rethinking ALS: the FUS about TDP-43. Cell 136, 1001-1004, doi: S0092-8674(09)00263-3 [pii]10.1016/j.cell.2009.03.006. |
| [4] | Da Cruz S & Cleveland DW (2011) Understanding the role of TDP-43 and FUS/TLS in ALS and beyond. Current Opinion in Neurobiology 21, 904-919, doi: 10.1016/j.conb.2011.05.029. |
| [5] | Taylor JP, Brown Jr RH & Cleveland DW (2016) Decoding ALS: from genes to mechanism. Nature 539, 197-206, doi: 10.1038/nature20413. |
| [6] | Van Langenhove T, van der Zee J, Sleegers K, Engelborghs S, Vandenberghe R, Gijselinck I, Van den Broeck M, Mattheijssens M, Peeters K, De Deyn PP, et al. (2010) Genetic contribution of FUS to frontotemporal lobar degeneration. Neurology 74, 366-371, doi: 74/5/366 [pii]10.1212/WNL.0b013e3181ccc732. |
| [7] | Broustal O, Camuzat A, Guillot-Noel L, Guy N, Millecamps S, Deffond D, Lacomblez L, Golfier V, Hannequin D, Salachas F, et al. (2010) FUS mutations in frontotemporal lobar degeneration with amyotrophic lateral sclerosis. J Alzheimers Dis 22, 765-769. |
| [8] | Martinelli I, Zucchi E, Pensato V, Gellera C, Traynor BJ, Gianferrari G, Chio A & Mandrioli J (2022) G507D mutation in FUS gene causes familial amyotrophic lateral sclerosis with a specific genotype-phenotype correlation. Neurobiology of aging 118, 124-128, doi: 10.1016/j.neurobiolaging.2022.05.006. |
| [9] | Zou H, Wang J-Y, Ma G-M, Xu M-M, Luo F, Zhang L & Wang W-Y (2022) The function of FUS in neurodevelopment revealed by the brain and spinal cord organoids. Molecular and Cellular Neuroscience 123, 103771, doi: DOI: 10.1016/j.mcn.2022.10377 |
| [10] | Korobeynikov VA, Lyashchenko AK, Blanco-Redondo B, Jafar-Nejad P & Shneider NA (2022) Antisense oligonucleotide silencing of FUS expression as a therapeutic approach in amyotrophic lateral sclerosis. Nature Medicine 28, 104-116, doi: 10.1038/s41591-021-01615-z. |
| [11] | Kurokawa R (2015) Long Noncoding RNAs. In, pp. 257. Springer. |
| [12] | Lipovich L, Tarca AL, Cai J, Jia H, Chugani HT, Sterner KN, Grossman LI, Uddin M, Hof PR, Sherwood CC, et al. (2014) Developmental changes in the transcriptome of human cerebral cortex tissue: long noncoding RNA transcripts. Cerebral cortex 24, 1451-1459, doi: 10.1093/cercor/bhs414. |
| [13] | Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, Guernec G, Martin D, Merkel A, Knowles DG, et al. (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome research 22, 1775-1789, doi: 10.1101/gr.132159.111. |
| [14] | Carninci P & Kasukawa T & Katayama S & Gough J & Frith MC & Maeda N & Oyama R & Ravasi T & Lenhard B & Wells C, et al. (2005) The transcriptional landscape of the mammalian genome. Science 309, 1559-1563, doi: 309/5740/1559 [pii]10.1126/science.1112014. |
| [15] | Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A, et al. (2009) Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A 106, 11667-11672, doi: 0904715106 [pii]10.1073/pnas.0904715106. |
| [16] | Necsulea A, Soumillon M, Warnefors M, Liechti A, Daish T, Zeller U, Baker JC, Grutzner F & Kaessmann H (2014) The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature 505, 635-640, doi: 10.1038/nature12943. |
| [17] | Chi KR (2016) Finding function in mystery transcripts. Nature 529, 423-425, doi: 10.1038/529423a. |
| [18] | Kurokawa R (2012) Generation of Functional Long Noncoding RNA Through Transcription and Natural Selection. In Regulatory RNAs, pp. 151-174. Springer. |
| [19] | Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, Tanzer A, Lagarde J, Lin W, Schlesinger F, et al. (2012) Landscape of transcription in human cells. Nature 489, 101-108, doi: 10.1038/nature11233. |
| [20] | Hon C-C, Ramilowski JA, Harshbarger J, Bertin N, Rackham OJL, Gough J, Denisenko E, Schmeier S, Poulsen TM, Severin J, et al. (2017) An atlas of human long non-coding RNAs with accurate 5′ ends. Nature 543, 199-204, doi: 10.1038/nature21374. |
| [21] | Kurokawa R (2011) Long noncoding RNA as a regulator for transcription. Prog Mol Subcell Biol 51, 29-41, doi: 10.1007/978-3-642-16502-3_2. |
| [22] | Kurokawa R (2011) Promoter-associated long noncoding RNAs repress transcription through a RNA binding protein TLS. Advances in experimental medicine and biology 722, 196-208, doi: 10.1007/978-1-4614-0332-6_12. |
| [23] | Kurokawa R (2015) Initiation of Transcription Generates Divergence of Long Noncoding RNAs. In Long Noncoding RNAs, pp. 69-91. Springer. |
| [24] | Aguilar R, Spencer KB, Kesner B, Rizvi NF, Badmalia MD, Mrozowich T, Mortison JD, Rivera C, Smith GF, Burchard J, et al. (2022) Targeting Xist with compounds that disrupt RNA structure and X inactivation. Nature 604, 160-166, doi: 10.1038/s41586-022-04537-z. |
| [25] | Yang F, Tanasa B, Micheletti R, Ohgi KA, Aggarwal AK & Rosenfeld MG (2021) Shape of promoter antisense RNAs regulates ligand-induced transcription activation. Nature 595, 444-449, doi: 10.1038/s41586-021-03589-x. |
| [26] | Allou L, Balzano S, Magg A, Quinodoz M, Royer-Bertrand B, Schöpflin R, Chan W-L, Speck-Martins CE, Carvalho DR, Farage L, et al. (2021) Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator. Nature 592, 93-98, doi: 10.1038/s41586-021-03208-9. |
| [27] | Kurokawa R, Rosenfeld MG & Glass CK (2009) Transcriptional regulation through noncoding RNAs and epigenetic modifications. RNA Biol 6, 233-236, doi: 8329 [pii]. |
| [28] | Cech TR & Steitz JA (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-94, doi: 10.1016/j.cell.2014.03.008. |
| [29] | Liu SJ, Horlbeck MA, Cho SW, Birk HS, Malatesta M, He D, Attenello FJ, Villalta JE, Cho MY, Chen Y, et al. (2017) CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111, doi: 10.1126/science.aah7111. |
| [30] | Cai Z, Cao C, Ji L, Ye R, Wang D, Xia C, Wang S, Du Z, Hu N, Yu X, et al. (2020) RIC-seq for global in situ profiling of RNA–RNA spatial interactions. Nature 582, 432-437, doi: 10.1038/s41586-020-2249-1. |
| [31] | Elguindy MM & Mendell JT (2021) NORAD-induced Pumilio phase separation is required for genome stability. Nature 595, 303-308, doi: 10.1038/s41586-021-03633-w. |
| [32] | Quinodoz SA, Jachowicz JW, Bhat P, Ollikainen N, Banerjee AK, Goronzy IN, Blanco MR, Chovanec P, Chow A, Markaki Y, et al. (2021) RNA promotes the formation of spatial compartments in the nucleus. Cell 184, 5775-5790.e5730, doi: 10.1016/j.cell.2021.10.014. |
| [33] | Chong PA, Vernon RM & Forman-Kay JD (2018) RGG/RG Motif Regions in RNA Binding and Phase Separation. J Mol Biol 430, 4650-4665, doi: 10.1016/j.jmb.2018.06.014. |
| [34] | Corley M, Burns MC & Yeo GW (2020) How RNA-Binding Proteins Interact with RNA: Molecules and Mechanisms. Molecular Cell 78, 9-29, doi: 10.1016/j.molcel.2020.03.011. |
| [35] | Lunde BM, Moore C & Varani G (2007) RNA-binding proteins: modular design for efficient function. Nature reviews Molecular cell biology 8, 479-490, doi: 10.1038/nrm2178. |
| [36] | Clery A, Blatter M & Allain FH (2008) RNA recognition motifs: boring? Not quite. Current opinion in structural biology 18, 290-298, doi: 10.1016/j.sbi.2008.04.002. |
| [37] | Linder P & Jankowsky E (2011) From unwinding to clamping - the DEAD box RNA helicase family. Nature reviews Molecular cell biology 12, 505-516, doi: 10.1038/nrm3154. |
| [38] | Ramakrishnan V (2014) The ribosome emerges from a black box. Cell 159, 979-984, doi: 10.1016/j.cell.2014.10.052. |
| [39] | Steitz TA (2008) A structural understanding of the dynamic ribosome machine. Nature reviews Molecular cell biology 9, 242-253, doi: 10.1038/nrm2352. |
| [40] | Behrmann E, Loerke J, Budkevich TV, Yamamoto K, Schmidt A, Penczek PA, Vos MR, Burger J, Mielke T, Scheerer P, et al. (2015) Structural snapshots of actively translating human ribosomes. Cell 161, 845-857, doi: 10.1016/j.cell.2015.03.052. |
| [41] | Papasaikas P & Valcarcel J (2016) The Spliceosome: The Ultimate RNA Chaperone and Sculptor. Trends in biochemical sciences 41, 33-45, doi: 10.1016/j.tibs.2015.11.003. |
| [42] | Plaschka C, Lin PC & Nagai K (2017) Structure of a pre-catalytic spliceosome. Nature 546, 617-621, doi: 10.1038/nature22799. |
| [43] | Beckmann BM, Horos R, Fischer B, Castello A, Eichelbaum K, Alleaume A-M, Schwarzl T, Curk T, Foehr S, Huber W, et al. (2015) The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs. Nature Communications 6, 10127, doi: 10.1038/ncomms10127. |
| [44] | Xu Y, Huangyang P, Wang Y, Xue L, Devericks E, Nguyen HG, Yu X, Oses-Prieto JA, Burlingame AL, Miglani S, et al. (2021) ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance. Cell 184, 5215-5229.e5217, doi: 10.1016/j.cell.2021.08.036. |
| [45] | Wright PE & Dyson HJ (1999) Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 293, 321-331, doi: 10.1006/jmbi.1999.3110. |
| [46] | Wright PE & Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nature reviews Molecular cell biology 16, 18-29, doi: 10.1038/nrm3920. |
| [47] | Han TW, Kato M, Xie S, Wu LC, Mirzaei H, Pei J, Chen M, Xie Y, Allen J, Xiao G, et al. (2012) Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell 149, 768-779, doi: 10.1016/j.cell.2012.04.016. |
| [48] | Ozdilek BA, Thompson VF, Ahmed NS, White CI, Batey RT & Schwartz JC (2017) Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding. Nucleic acids research 45, 7984-7996, doi: 10.1093/nar/gkx460. |
| [49] | Lin Y, Currie SL & Rosen MK (2017) Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs. Journal of Biological Chemistry 292, 19110-19120, doi: 10.1074/jbc.M117.800466. |
| [50] | Balcerak A, Trebinska-Stryjewska A, Konopinski R, Wakula M & Grzybowska EA (2019) RNA-protein interactions: disorder, moonlighting and junk contribute to eukaryotic complexity. Open Biol 9, 190096, doi: 10.1098/rsob.190096. |
| [51] | Zeke A, Schád É, Horváth T, Abukhairan R, Szabó B & Tantos A (2022) Deep structural insights into RNA-binding disordered protein regions. WIREs RNA 13, e1714, doi: 10.1002/wrna.1714. |
| [52] | Castello A, Fischer B, Frese CK, Horos R, Alleaume AM, Foehr S, Curk T, Krijgsveld J & Hentze MW (2016) Comprehensive Identification of RNA-Binding Domains in Human Cells. Molecular Cell 63, 696-710, doi: 10.1016/j.molcel.2016.06.029. |
| [53] | Kwon SC, Yi H, Eichelbaum K, Fohr S, Fischer B, You KT, Castello A, Krijgsveld J, Hentze MW & Kim VN (2013) The RNA-binding protein repertoire of embryonic stem cells. Nature structural & molecular biology 20, 1122-1130, doi: 10.1038/nsmb.2638. |
| [54] | Liepelt A, Naarmann-de Vries IS, Simons N, Eichelbaum K, Fohr S, Archer SK, Castello A, Usadel B, Krijgsveld J, Preiss T, et al. (2016) Identification of RNA-binding Proteins in Macrophages by Interactome Capture. Molecular & cellular proteomics: MCP 15, 2699-2714, doi: 10.1074/mcp.M115.056564. |
| [55] | Loughlin FE, Lukavsky PJ, Kazeeva T, Reber S, Hock EM, Colombo M, Von Schroetter C, Pauli P, Clery A, Muhlemann O, et al. (2019) The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity. Molecular cell 73, 490-504 e496, doi: 10.1016/j.molcel.2018.11.012. |
| [56] | Wang X, Arai S, Song X, Reichart D, Du K, Pascual G, Tempst P, Rosenfeld MG, Glass CK & Kurokawa R (2008) Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature 454, 126-130, doi: nature06992 [pii]10.1038/nature06992. |
| [57] | Du K, Arai S, Kawamura T, Matsushita A & Kurokawa R (2011) TLS and PRMT1 synergistically coactivate transcription at the survivin promoter through TLS arginine methylation. Biochem Biophys Res Commun 404, 991-996, doi: S0006-291X(10)02335-1 [pii]10.1016/j.bbrc.2010.12.097. |
| [58] | Cui W, Yoneda R, Ueda N & Kurokawa R (2018) Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity. J Biol Chem 293, 10937-10948, doi: 10.1074/jbc.RA117.000598. |
| [59] | Ueda N & Kurokawa R (2018) Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups. Biomedical Sciences 4, 24-31, doi: 10.11648/j.bs.20180403.11 |
| [60] | Yoneda R, Suzuki S, Mashima T, Kondo K, Nagata T, Katahira M & Kurokawa R (2016) The binding specificity of Translocated in LipoSarcoma/FUsed in Sarcoma with lncRNA transcribed from the promoter region of cyclin D1. Cell & bioscience 6, 4, doi: 10.1186/s13578-016-0068-8. |
| [61] | Yoneda R, Ueda N, Uranishi K, Hirasaki M & Kurokawa R (2020) Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m (6) A modification. J Biol Chem 295, 5626-5639, doi: 10.1074/jbc.RA119.011556. |
| [62] | Yoneda R, Ueda N & Kurokawa R (2021) m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress. International Journal of Molecular Sciences 22, 11014, doi: 10.3390/ijms222011014 |
| [63] | Hamad N, Yoneda R, So M, Kurokawa R, Nagata T & Katahira M (2021) Non-coding RNA suppresses FUS aggregation caused by mechanistic shear stress on pipetting in a sequence-dependent manner. Sci Rep 11, 9523, doi: 10.1038/s41598-021-89075-w. |
| [64] | Wang X, Schwartz JC & Cech TR (2015) Nucleic acid-binding specificity of human FUS protein. Nucleic Acids Research 43, 7535-7543, doi: 10.1093/nar/gkv679. |
| [65] | Tejedor AR, Sanchez-Burgos I, Estevez-Espinosa M, Garaizar A, Collepardo-Guevara R, Ramirez J & Espinosa JR (2022) Protein structural transitions critically transform the network connectivity and viscoelasticity of RNA-binding protein condensates but RNA can prevent it. Nature Communications 13, 5717, doi: 10.1038/s41467-022-32874-0. |
| [66] | Du S, Wu S, Feng X, Wang B, Xia S, Liang L, Zhang L, Govindarajalu G, Bunk A, Kadakia F, et al. (2022) A nerve injury–specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression. The Journal of Clinical Investigation 132, doi: 10.1172/jci153563. |
| [67] | Luo X-J, He M-M, Liu J, Zheng J-B, Wu Q-N, Chen Y-X, Meng Q, Luo K-J, Chen D-L, Xu R-H, et al. (2022) LncRNA TMPO-AS1 promotes esophageal squamous cell carcinoma progression by forming biomolecular condensates with FUS and p300 to regulate TMPO transcription. Experimental & Molecular Medicine 54, 834-847, doi: 10.1038/s12276-022-00791-3. |
| [68] | Xing C-Y, Zhang Y-Z, Hu W & Zhao L-Y (2022) LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression. Cellular and Molecular Life Sciences 79, 382, doi: 10.1007/s00018-022-04376-1. |
| [69] | Roeder RG (1991) The complexities of eukaryotic transcription initiation: regulation of preinitiation complex assembly. Trends Biochem Sci 16, 402-408. |
| [70] | Roeder RG (2003) Lasker Basic Medical Research Award. The eukaryotic transcriptional machinery: complexities and mechanisms unforeseen. Nature medicine 9, 1239-1244, doi: 10.1038/nm938. |
| [71] | Glass CK & Rosenfeld MG (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14, 121-141. |
| [72] | Rosenfeld MG, Lunyak VV & Glass CK (2006) Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. Genes & development 20, 1405-1428. |
| [73] | Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR & Sigler PB (1991) Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352, 497-505, doi: 10.1038/352497a0. |
| [74] | Glass CK, Rosenfeld MG, Rose DW, Kurokawa R, Kamei Y, Xu L, Torchia J, Ogliastro MH & Westin S (1997) Mechanisms of transcriptional activation by retinoic acid receptors. Biochem Soc Trans 25, 602-605. |
| [75] | Kurokawa R, Soderstrom M, Horlein A, Halachmi S, Brown M, Rosenfeld MG & Glass CK (1995) Polarity-specific activities of retinoic acid receptors determined by a co-repressor. Nature 377, 451-454, doi: 10.1038/377451a0. |
| [76] | Kurokawa R, Yu VC, Naar A, Kyakumoto S, Han Z, Silverman S, Rosenfeld MG & Glass CK (1993) Differential orientations of the DNA-binding domain and carboxy-terminal dimerization interface regulate binding site selection by nuclear receptor heterodimers. Genes Dev 7, 1423-1435. |
| [77] | Dreyfuss G, Kim VN & Kataoka N (2002) Messenger-RNA-binding proteins and the messages they carry. Nature reviews Molecular cell biology 3, 195-205, doi: 10.1038/nrm760. |
| [78] | Hudson WH & Ortlund EA (2014) The structure, function and evolution of proteins that bind DNA and RNA. Nature reviews Molecular cell biology 15, 749-760, doi: 10.1038/nrm3884. |
| [79] | Hentze MW, Castello A, Schwarzl T & Preiss T (2018) A brave new world of RNA-binding proteins. Nature reviews Molecular cell biology 19, 327-341, doi: 10.1038/nrm.2017.130. |
| [80] | Hasegawa Y, Brockdorff N, Kawano S, Tsutui K & Nakagawa S (2010) The matrix protein hnRNP U is required for chromosomal localization of Xist RNA. Developmental cell 19, 469-476, doi: 10.1016/j.devcel.2010.08.006. |
| [81] | Lambert N, Robertson A, Jangi M, McGeary S, Sharp PA & Burge CB (2014) RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins. Molecular Cell 54, 887-900, doi: 10.1016/j.molcel.2014.04.016. |
| [82] | Zhou Q, Kunder N, De la Paz JA, Lasley AE, Bhat VD, Morcos F & Campbell ZT (2018) Global pairwise RNA interaction landscapes reveal core features of protein recognition. Nature communications 9, 2511, doi: 10.1038/s41467-018-04729-0. |
| [83] | Jankowsky E & Harris ME (2015) Specificity and nonspecificity in RNA-protein interactions. Nature reviews Molecular cell biology 16, 533-544, doi: 10.1038/nrm4032. |
| [84] | Kim MK & Nikodem VM (1999) hnRNP U Inhibits Carboxy-Terminal Domain Phosphorylation by TFIIH and Represses RNA Polymerase II Elongation. Molecular and Cellular Biology 19, 6833-6844. |
| [85] | Berglund FM & Clarke PR (2009) hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks. Biochemical and biophysical research communications 381, 59-64, doi: 10.1016/j.bbrc.2009.02.019. |
| [86] | Polo SE, Blackford AN, Chapman JR, Baskcomb L, Gravel S, Rusch A, Thomas A, Blundred R, Smith P, Kzhyshkowska J, et al. (2012) Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair. Molecular Cell 45, 505-516, doi: 10.1016/j.molcel.2011.12.035. |
| [87] | Jiang H, Wang Y, Ai M, Wang H, Duan Z, Wang H, Zhao L, Yu J, Ding Y & Wang S (2017) Long noncoding RNA CRNDE stabilized by hnRNPUL2 accelerates cell proliferation and migration in colorectal carcinoma via activating Ras/MAPK signaling pathways. Cell Death & Disease 8, e2862-e2862, doi: 10.1038/cddis.2017.258. |
| [88] | Zhou X & Feliciano P & Shu C & Wang T & Astrovskaya I & Hall JB & Obiajulu JU & Wright JR & Murali SC & Xu SX, et al. (2022) Integrating de novo and inherited variants in 42,607 autism cases identifies mutations in new moderate-risk genes. Nature Genetics 54, 1305-1319, doi: 10.1038/s41588-022-01148-2. |
| [89] | Lane AN, Chaires JB, Gray RD & Trent JO (2008) Stability and kinetics of G-quadruplex structures. Nucleic Acids Research 36, 5482-5515, doi: 10.1093/nar/gkn517. |
| [90] | Booy EP, Meier M, Okun N, Novakowski SK, Xiong S, Stetefeld J & McKenna SA (2012) The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary. Nucleic Acids Research 40, 4110-4124, doi: 10.1093/nar/gkr1306. |
| [91] | Rhodes D & Lipps HJ (2015) G-quadruplexes and their regulatory roles in biology. Nucleic Acids Research 43, 8627-8637, doi: 10.1093/nar/gkv862. |
| [92] | Takahama K & Oyoshi T (2013) Specific binding of modified RGG domain in TLS/FUS to G-quadruplex RNA: tyrosines in RGG domain recognize 2'-OH of the riboses of loops in G-quadruplex. Journal of the American Chemical Society 135, 18016-18019, doi: 10.1021/ja4086929. |
| [93] | Takahama K, Takada A, Tada S, Shimizu M, Sayama K, Kurokawa R & Oyoshi T (2013) Regulation of telomere length by G-quadruplex telomere DNA- and TERRA-binding protein TLS/FUS. Chemistry & biology 20, 341-350, doi: 10.1016/j.chembiol.2013.02.013. |
| [94] | Li W, Notani D & Rosenfeld MG (2016) Enhancers as non-coding RNA transcription units: recent insights and future perspectives. Nature reviews Genetics 17, 207-223, doi: 10.1038/nrg.2016.4. |
| [95] | Song C, Hotz-Wagenblatt A, Voit R & Grummt I (2017) SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability. Genes & development 31, 1370-1381, doi: 10.1101/gad.300624.117. |
| [96] | Hua JT, Ahmed M, Guo H, Zhang Y, Chen S, Soares F, Lu J, Zhou S, Wang M, Li H, et al. (2018) Risk SNP-Mediated Promoter-Enhancer Switching Drives Prostate Cancer through lncRNA PCAT19. Cell 174, 564-575 e518, doi: 10.1016/j.cell.2018.06.014. |
| [97] | Hoell JI, Larsson E, Runge S, Nusbaum JD, Duggimpudi S, Farazi TA, Hafner M, Borkhardt A, Sander C & Tuschl T (2011) RNA targets of wild-type and mutant FET family proteins. Nature structural & molecular biology 18, 1428-1431, doi: 10.1038/nsmb.2163. |
| [98] | Kato M, Han TW, Xie S, Shi K, Du X, Wu LC, Mirzaei H, Goldsmith EJ, Longgood J, Pei J, et al. (2012) Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753-767, doi: 10.1016/j.cell.2012.04.017. |
| [99] | Chiang W-C, Lee M-H, Chen T-C & Huang J-r (2020) Interactions between the Intrinsically Disordered Regions of hnRNP-A2 and TDP-43 Accelerate TDP-43′s Conformational Transition. International Journal of Molecular Sciences 21, 5930, doi: doi.org/10.3390/ijms21165930. |
| [100] | Carter GC, Hsiung C-H, Simpson L, Yang H & Zhang X (2021) N-terminal Domain of TDP43 Enhances Liquid-Liquid Phase Separation of Globular Proteins. J Mol Biol 433, 166948, doi: https://doi.org/10.1016/j.jmb.2021.166948. |
APA Style
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
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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Download@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://article.sciencepublishinggroup.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}
}
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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