Volume 11 Issue 9
Aug.  2020
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Bohong Chen, Shengcheng Deng, Tianyu Ge, Miaoman Ye, Jianping Yu, Song Lin, Wenbin Ma, Zhou Songyang. Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in[J]. Protein&Cell, 2020, 11(9): 641-660. doi: 10.1007/s13238-020-00706-w
Citation: Bohong Chen, Shengcheng Deng, Tianyu Ge, Miaoman Ye, Jianping Yu, Song Lin, Wenbin Ma, Zhou Songyang. Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in[J]. Protein&Cell, 2020, 11(9): 641-660. doi: 10.1007/s13238-020-00706-w

Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in

doi: 10.1007/s13238-020-00706-w
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We would like to thank Dr. Dan Liu for helping with the manuscript. This work was supported by the National Key Research and Development Program of China (2017YFA0102801, 2018YFA0107003), National Natural Science Foundation of China (91640119, 91749113, 31570827, 31871479 and 31930058), Natural Science Foundation of Guangdong Province (2017A030313116) and the China Postdoctoral Science Foundation (2018M631021).

  • Received Date: 2019-11-20
  • Rev Recd Date: 2020-02-19
  • In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.
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  • [1]
    Bae S, Kweon J, Kim HS, Kim J (2014) Microhomology-based choice of Cas9 nuclease target sites. Nat Methods 11:705-706
    [2]
    Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, Aumonier S, Gotthard G, Royant A, Hink MA et al(2017) mScarlet:a bright monomeric red fluorescent protein for cellular imaging. Nat Methods 14:53-56
    [3]
    Cao M, Zhao J, Hu G (2019) Genome-wide methods for investigating long noncoding RNAs. Biomed Pharmacother 111:395-401
    [4]
    Chen L (2016) Linking long noncoding RNA localization and function. Trends Biochem Sci 41:761-772
    [5]
    Chen L, Carmichael GG (2009) Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells:functional role of a nuclear noncoding RNA. Mol Cell 35:467-478
    [6]
    Chujo T, Yamazaki T, Kawaguchi T, Kurosaka S, Takumi T, Nakagawa S, Hirose T (2017) Unusual semi-extractability as a hallmark of nuclear body-associated architectural noncoding RNAs. EMBO J 36:1447-1462
    [7]
    Clemson CM, Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess A, Lawrence JB (2009) An architectural role for a nuclear noncoding RNA:NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell 33:717-726
    [8]
    Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing with CRISPR-Cas13. Science (New York, N.Y.) 358:1019-1027
    [9]
    Delacôte F, Deriano L, Lambert S, Bertrand P, Saintigny Y, Lopez BS (2007) Chronic exposure to sublethal doses of radiation mimetic ZeocinTM selects for clones deficient in homologous recombination. Mutat Res 615:125-133
    [10]
    Deng T, Huang Y, Weng K, Lin S, Li Y, Shi G, Chen Y, Huang J, Liu D, Ma W et al (2019) TOE1 acts as a 3'exonuclease for telomerase RNA and regulates telomere maintenance. Nucleic Acids Res 47:391-405
    [11]
    Derrien T, Guigó R, Johnson R (2012) The long non-coding RNAs:a new (P)layer in the "Dark Matter". Front Genet 2:107
    [12]
    Evans JR, Feng FY, Chinnaiyan AM (2016) The bright side of dark matter:lncRNAs in cancer. J Clin Invest 126:2775-2782
    [13]
    Fox AH, Bond CS, Lamond AI (2005) P54nrb forms a heterodimer with PSP1 that localizes to paraspeckles in an RNA-dependent manner. Mol Biol Cell 16:5304-5315
    [14]
    Fox AH, Lam YW, Leung AK, Lyon CE, Andersen J, Mann M, Lamond AI (2002) Paraspeckles:a novel nuclear domain. Curr Biol 12:13-25
    [15]
    Fox AH, Nakagawa S, Hirose T, Bond CS (2018) Paraspeckles:where long noncoding RNA meets phase separation. Trends Biochem Sci 43:124-135
    [16]
    Fujimoto A, Furuta M, Totoki Y, Tsunoda T, Kato M, Shiraishi Y, Tanaka H, Taniguchi H, Kawakami Y, Ueno M et al (2016) Wholegenome mutational landscape and characterization of noncoding and structural mutations in liver cancer. Nat Genet 48:500-509
    [17]
    Fusco D, Accornero N, Lavoie B, Shenoy SM, Blanchard J, Singer RH, Bertrand E (2003) Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. CURR Biol 13:161-167
    [18]
    George L, Indig FE, Abdelmohsen K, Gorospe M (2018) Intracellular RNA-tracking methods. Open Biol 8:180104
    [19]
    Hirose T, Virnicchi G, Tanigawa A, Naganuma T, Li R, Kimura H, Yokoi T, Nakagawa S, Benard M, Fox AH et al (2014) NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies. Mol Biol Cell 25:169-183
    [20]
    Hu S, Yao R, Chen L (2016) Shedding light on paraspeckle structure by super-resolution microscopy. J Cell Biol 214:789-791
    [21]
    Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A (2007) A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics 8:39
    [22]
    Imamura K, Imamachi N, Akizuki G, Kumakura M, Kawaguchi A, Nagata K, Kato A, Kawaguchi Y, Sato H, Yoneda M et al (2014) Long noncoding RNA NEAT1-dependent SFPQ relocation from promoter region to paraspeckle mediates IL8 expression upon immune stimuli. Mol Cell 53:393-406
    [23]
    Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging technology. Nat Methods 8:S12-S19
    [24]
    Jandura A, Krause HM (2017) The new RNA world:growing evidence for long noncoding RNA functionality. Trends Genet 33:665-676
    [25]
    Kawakami J, Sugimoto N, Tokitoh H, Tanabe Y (2006) A novel stable RNA pentaloop that interacts specifically with a motif peptide of lambda-N protein. Nucleosides Nucleotides Nucleic Acids 25:397-416
    [26]
    Kim S, Kim D, Cho SW, Kim J, Kim JS (2014) Highly efficient RNAguided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012-1019
    [27]
    Kim SH, Vieira M, Kim H, Kesawat MS, Park HY (2019) MS2 labeling of endogenous beta-actin mRNA does not result in stabilization of degradation intermediates. Mol Cells 42:356-362
    [28]
    Knott GJ, Bond CS, Fox AH (2016) The DBHS proteins SFPQ, NONO and PSPC1:a multipurpose molecular scaffold. Nucleic Acids Res 44:3989-4004
    [29]
    Kopp F, Mendell JT (2018) Functional classification and experimental dissection of long noncoding RNAs. Cell 172:393-407
    [30]
    Kostyrko K, Mermod N (2016) Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms. Nucleic Acids Res 44:e56
    [31]
    Lanzós A, Carlevaro-Fita J, Mularoni L, Reverter F, Palumbo E, Guigó R, Johnson R (2017) Discovery of cancer driver long noncoding RNAs across 1112 tumour genomes:new candidates and distinguishing features. Sci Rep UK 7:1-16
    [32]
    Lee M, Sadowska A, Bekere I, Ho D, Gully BS, Lu Y, Iyer KS, Trewhella J, Fox AH, Bond CS (2015) The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation. Nucleic Acids Res 43:3826-3840
    [33]
    Lee O, Kim H, He Q, Baek HJ, Yang D, Chen L, Liang J, Chae HK, Safari A, Liu D et al (2011) Genome-wide YFP fluorescence complementation screen identifies new regulators for telomere signaling in human cells. Mol Cell Proteomics 10:M110-M1628
    [34]
    Levsky JM, Singer RH (2003) Fluorescence in situ hybridization:past, present and future. J CELL SCI 116:2833-2838
    [35]
    Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011) A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods 8:165-170
    [36]
    Liu S, Zhu J, Jiang T, Zhong Y, Tie Y, Wu Y, Zheng X, Jin Y, Fu H (2015) Identification of lncRNA MEG3 binding protein using MS2-tagged RNA affinity purification and mass spectrometry. Appl Biochem Biotech 176:1834-1845
    [37]
    Ma L, Bajic VB, Zhang Z (2014) On the classification of long noncoding RNAs. RNA Biol 10:924-933
    [38]
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823-826
    [39]
    Marchese FP, Raimondi I, Huarte M (2017) The multidimensional mechanisms of long noncoding RNA function. Genome Biol 18:206
    [40]
    Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP:a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401-W407
    [41]
    Munschauer M, Nguyen CT, Sirokman K, Hartigan CR, Hogstrom L, Engreitz JM, Ulirsch JC, Fulco CP, Subramanian V, Chen J et al(2018) The NORAD lncRNA assembles a topoisomerase complex critical for genome stability. Nature 561:132-136
    [42]
    Naganuma T, Nakagawa S, Tanigawa A, Sasaki YF, Goshima N, Hirose T (2012) Alternative 3'-end processing of long noncoding RNA initiates construction of nuclear paraspeckles. EMBO J 31:4020-4034
    [43]
    Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T et al (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 5:1-8
    [44]
    Nakagawa S, Shimada M, Yanaka K, Mito M, Arai T, Takahashi E, Fujita Y, Fujimori T, Standaert L, Marine JC et al (2014) The lncRNA Neat1 is required for corpus luteum formation and the establishment of pregnancy in a subpopulation of mice. Development 141:4618-4627
    [45]
    Nelles DA, Fang MY, O Connell MR, Xu JL, Markmiller SJ, Doudna JA, Yeo GW (2016) Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165:488-496
    [46]
    Nguyen VT, Kiss T, Michels AA, Bensaude O (2001) 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature 414:322-325
    [47]
    Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, Choi VM, Gopalan SM, Lou JF, Li J et al (2010) Zincfinger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res 38:152
    [48]
    Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, Singer RH (2014) Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science 343:422-424
    [49]
    Perry RP, Kelley DE (1970) Inhibition of RNA synthesis by actinomycin D:characteristic dose-response of different RNA species. J Cell Physiol 76:127-139
    [50]
    Qin P, Parlak M, Kuscu C, Bandaria J, Mir M, Szlachta K, Singh R, Darzacq X, Yildiz A, Adli M (2017) Live cell imaging of low-and non-repetitive chromosome loci using CRISPR-Cas9. Nat Commun 8:1-10
    [51]
    Ramanathan M, Majzoub K, Rao DS, Neela PH, Zarnegar BJ, Mondal S, Roth JG, Gai H, Kovalski JR, Siprashvili Z et al (2018) RNA-protein interaction detection in living cells. Nat Methods 15:207-212
    [52]
    Rau K, Rentmeister A (2016) CRISPR/Cas9:a new tool for RNA imaging in live cells. ChemBioChem 17:1682-1684
    [53]
    Roots R, Smith KC (1976) Effects of actinomycin D on cell cycle kinetics and the DNA of Chinese hamster and mouse mammary tumor cells cultivated in vitro. Cancer Res 36:3654-3658
    [54]
    Sasaki YTF, Ideue T, Sano M, Mituyama T, Hirose T (2009) MEN[epsilon]/[beta] noncoding RNAs are essential for structural integrity of nuclear paraspeckles. Proc Natl Acad Sci USA 106:2525
    [55]
    Shao S, Zhang W, Hu H, Xue B, Qin J, Sun C, Sun Y, Wei W, Sun Y (2016) Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res 44:e86
    [56]
    Shelkovnikova TA, Robinson HK, Troakes C, Ninkina N, Buchman VL (2014) Compromised paraspeckle formation as a pathogenic factor in FUSopathies. Hum Mol Genet 23:2298-2312
    [57]
    Sobell HM (1985) Actinomycin and DNA Transcription. Proc Natl Acad Sci USA 82:5328-5331
    [58]
    Spille JH, Hecht M, Grube V, Cho WK, Lee C, Cisse II (2019) A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells. Methods 153:35-45
    [59]
    Standaert L, Adriaens C, Radaelli E, Van Keymeulen A, Blanpain C, Hirose T, Nakagawa S, Marine J (2014) The long noncoding RNA Neat1 is required for mammary gland development and lactation. RNA 20:1844-1849
    [60]
    Sunwoo H, Dinger ME, Wilusz JE, Amaral PP, Mattick JS, Spector DL (2008) MEN/nuclear-retained non-coding RNAs are upregulated upon muscle differentiation and are essential components of paraspeckles. Genome Res 19:347-359
    [61]
    Taleei R, Nikjoo H (2013) Biochemical DSB-repair model for mammalian cells in G1 and early S phases of the cell cycle. Mutat Res 756:206-212
    [62]
    Tasan I, Sustackova G, Zhang L, Kim J, Sivaguru M, HamediRad M, Wang Y, Genova J, Ma J, Belmont AS et al (2018) CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci. Nucleic Acids Res 46:e100
    [63]
    Tsai BP, Wang X, Huang L, Waterman ML (2011) Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach. Mol Cell Proteomics 10:M110-M7385
    [64]
    Tutucci E, Vera M, Biswas J, Garcia J, Parker R, Singer RH (2018a) An improved MS2 system for accurate reporting of the mRNA life cycle. Nat Methods 15:81-89
    [65]
    Tutucci E, Vera M, Singer RH (2018b) Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system. Nat Protoc 13:2268-2296
    [66]
    Wang Y, Hu S, Wang M, Yao R, Wu D, Yang L, Chen L (2018) Genome-wide screening of NEAT1 regulators reveals crossregulation between paraspeckles and mitochondria. Nat Cell Biol 20:1145-1158
    [67]
    Wang Z, Fan P, Zhao Y, Zhang S, Lu J, Xie W, Jiang Y, Lei F, Xu N, Zhang Y (2017) NEAT1 modulates herpes simplex virus-1 replication by regulating viral gene transcription. Cell Mol Life Sci 74:1117-1131
    [68]
    Weinmann R, Raskas HJ, Roeder RG (1974) Role of DNAdependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection. Proc Natl Acad Sci USA 71:3426-3439
    [69]
    Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, Bodnar AG, Lichtsteiner S, Kim NW, Trager JB et al (1997) Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 17:498
    [70]
    West JA, Davis CP, Sunwoo H, Simon MD, Sadreyev RI, Wang PI, Tolstorukov MY, Kingston RE (2014) The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol Cell 55:791-802
    [71]
    West JA, Mito M, Kurosaka S, Takumi T, Tanegashima C, Chujo T, Yanaka K, Kingston RE, Hirose T, Bond C et al (2016) Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization. J Cell Biol 214:817-830
    [72]
    Wu B, Chao JA, Singer RH (2012) Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys J 102:2936-2944
    [73]
    Wu C, Li T, Farh L, Lin L, Lin T, Yu Y, Yen T, Chiang C, Chan N (2011) Structural basis of type II topoisomerase inhibition by the anticancer drug etoposide. Science 333:459-462
    [74]
    Xing Y, Yao R, Zhang Y, Guo C, Jiang S, Xu G, Dong R, Yang L, Chen L (2017) SLERT regulates DDX21 Rings associated with Pol I transcription. Cell 169:664-678
    [75]
    Yang LZ, Wang Y, Li SQ, Yao RW, Luan PF, Wu H, Carmichael GG, Chen LL (2019) Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems. Mol Cell 76:981-997
    [76]
    Yang Y, Wen L, Zhu H (2015) Unveiling the hidden function of long non-coding RNA by identifying its major partner-protein. Cell Biosci 5:59
    [77]
    Yao R, Wang Y, Chen L (2019) Cellular functions of long noncoding RNAs. Nat Cell Biol 21:542-551
    [78]
    Zhang Q, Chen CY, Yedavalli VS, Jeang KT (2013) NEAT1 long noncoding RNA and paraspeckle bodies modulate HIV-1 posttranscriptional expression. MBIO 4:e512-e596
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