Abstract
More than 90% of the human genome have been found to be transcribed and most of the transcripts are noncoding (nc) RNAs (Willingham et al., Science 309:1570–1573, 2005; ENCODE-consortium, Science 306:636–640, 2004; Carninci et al., Science 309:1559–1563, 2005; Bertone et al., Science 306:2242–2246, 2004). Studies on ncRNAs have been radically progressed mainly regarding microRNAs, piRNAs, siRNAs, and related small ncRNAs of which length are relatively short nucleotides (Fire et al., Nature 391:806–811, 1998; Filipowicz et al., Nat Rev Genet 9:102–114, 2008; Lau et al., Science 313:363–367, 2006; Brennecke et al., Science 322:1387–1392, 2008; Siomi and Siomi, Nature 457:396–404, 2009). These small RNAs play roles in regulation of translation and gene silencing while long ncRNAs with length more than 200 nucleotides have been emerging and turn out to be involved in regulation of transcription (Kapranov et al., Science 316:1484–1488, 2007; Ponting et al., Cell 136:629–641, 2009; Kurokawa et al., RNA Biol 6:233–236, 2009). Recently, we have identified novel, long ncRNAs bearing capability of repression of transcription (Wang et al., Nature 454:126–130, 2008).
RNA-binding protein, translocated in liposarcoma (TLS), binds CREB-binding protein CBP/adenovirus p300 and inhibits their histone acetyltransferase (HAT) activities (Wang et al., Nature 454:126–130, 2008). The HAT inhibitory activity of TLS requires specific binding of RNA. The systematic evolution of ligands by exponential enrichment experiments with randomized sequences revealed that TLS specifically recognizes RNA oligonucleotides containing GGUG as a consensus sequence although the GGUG sequence is not an absolute requirement for the TLS binding (Lerga et al., J Biol Chem 276:6807–6816, 2001). TLS is specifically recruited to the CBP/p300-associated binding sites of the cyclin D1 gene (CCND1) and the cyclin E1 gene (CCNE1) promoters (Wang et al., Nature 454:126–130, 2008; Impey et al., Cell 119:1041–1054, 2004). Our extensive exploration for naturally occurring RNA molecule that binds TLS has indicated that long ncRNAs (promoter-associated ncRNAs: pncRNAs) transcribed from the CCND1 promoter bind TLS and inhibit the HAT activities on the sites to repress the transcription of the CCND1 gene (Wang et al., Nature 454:126–130, 2008). We have optimized RT-PCR, chromatin immunoprecipitation, RNA immunoprecipitation, and RNA gel-shift assay in order to detect these pncRNAs. The methods that we have developed successfully identified these low-abundant, long ncRNAs and provide the data showing that the CCND1 pncRNAs bind TLS and induce its HAT inhibitory activity to repress the transcription of CCND1 gene upon genotoxic stress.
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Willingham AT, Orth AP, Batalov S, Peters EC, Wen BG, Aza-Blanc P, Hogenesch JB, Schultz PG (2005) A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science 3091570–1573.
ENCODE-consortium 2004 The ENCODE (ENCyclopedia Of DNA Elements) Project. Science 306 636–640.
Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, et al (2005) The transcriptional landscape of the mammalian genome. Science 309 1559–1563.
Bertone P, Stolc V, Royce TE, Rozowsky JS, Urban AE, Zhu X, Rinn JL, Tongprasit W, Samanta M, Weissman S, Gerstein M, Snyder M (2004) Global identification of human transcribed sequences with genome tiling arrays. Science 306 2242–2246.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391 806–811.
Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9 102–114.
Lau NC, Seto AG, Kim J, Kuramochi-Miyagawa S, Nakano T, Bartel DP, Kingston RE (2006) Characterization of the piRNA complex from rat testes. Science 313,363–367.
Brennecke J, Malone CD, Aravin AA, Sachidanandam R, Stark A, Hannon GJ (2008) An epigenetic role for maternally inherited piRNAs in transposon silencing. Science 322 1387–1392.
Siomi H, Siomi MC (2009) On the road to reading the RNA-interference code. Nature 457, 396–404.
Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT, Stadler PF, Hertel J, Hackermuller J, Hofacker IL, Bell I, Cheung E, Drenkow J, Dumais E, Patel S, Helt G, Ganesh M, Ghosh S, Piccolboni A, Sementchenko V, Tammana H, Gingeras TR (2007) RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316, 1484–1488.
Ponting CP, Oliver PL, Reik W (2009) Evolution and functions of long noncoding RNAs. Cell 136, 629–641.
Kurokawa R, Rosenfeld MG, Glass CK (2009) Transcriptional regulation through noncoding RNAs and epigenetic modifications. RNA Biol 6 233–236.
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.
Lerga A, Hallier M, Delva L, Orvain C, Gallais I, Marie J, Moreau-Gachelin F (2001) Identification of an RNA binding specificity for the potential splicing factor TLS. J Biol Chem 276, 6807–6816.
Impey S, McCorkle SR, Cha-Molstad H, Dwyer JM, Yochum GS, Boss JM, McWeeney S, Dunn JJ, Mandel G, Goodman RH (2004) Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119 1041–1054.
Glass CK, Rosenfeld MG (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14 121–141.
Murata T, Kurokawa R, Krones A, Tatsumi K, Ishii M, Taki T, Masuno M, Ohashi H, Yanagisawa M, Rosenfeld MG, Glass CK, Hayashi Y (2001) Defect of histone acetyltransferase activity of the nuclear transcriptional coactivator CBP in Rubinstein-Taybi syndrome. Hum Mol Genet 10 1071–1076.
Steffan JS, Bodai L, Pallos J, Poelman M, McCampbell A, Apostol BL, Kazantsev A, Schmidt E, Zhu YZ, Greenwald M, Kurokawa R, Housman DE, Jackson GR, Marsh JL, Thompson LM (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413 39–743.
Crozat A, Aman P, Mandahl N, Ron D (1993) Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature 363 640–644.
Kuroda M, Sok J, Webb L, Baechtold H, Urano F, Yin Y, Chung P, de Rooij DG, Akhmedov A, Ashley T, Ron D (2000) Male sterility and enhanced radiation sensitivity in TLS(−/−) mice. EMBO J 19 453–462.
Hicks GG, Singh N, Nashabi A, Mai S, Bozek G, Klewes L, Arapovic D, White EK, Koury MJ, Oltz EM, Van Kaer L, Ruley HE (2000) Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death. Nat Genet 24 175–179.
Al-Chalabi A, Leigh PN (2000) Recent advances in amyotrophic lateral sclerosis. Curr Opin Neurol 13 397–405.
Boillee S, Vande Velde C, Cleveland DW (2006) ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52 39–59.
Pasinelli P, Brown RH (2006) Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat Rev Neurosci 7 710–723.
Rosen DR (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 364 362.
Kwiatkowski TJ, Jr., Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T, Valdmanis P, Rouleau GA, Hosler BA, Cortelli P, de Jong PJ, Yoshinaga Y, Haines JL, Pericak-Vance MA, Yan J, Ticozzi N, Siddique T, McKenna-Yasek D, Sapp PC, Horvitz HR, Landers JE, Brown RH, Jr. (2009) Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323 1205–1208.
Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo JM, Miller CC, Shaw CE (2009) Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323 1208–1211.
Acknowledgments
The authors thank Ms. R. Tanji for preparation of the manuscript, and Dr. C.K. Glass and Dr. M.G. Rosenfeld for critical discussion. This work was supported by Takeda Science Foundation, the Naito foundations, Astellas Foundation for Research on Metabolic Disorders Foundation, and also Grant-in-Aid for Scientific Research (B: nos22390057) and Grant-in-aid for “Support Project of Strategic Research Center in Private Universities” from the Ministry of Education, Culture, Sports, Science and Technology to Saitama Medical University Research Center for Genomic Medicine.
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Song, X., Wang, X., Arai, S., Kurokawa, R. (2012). Promoter-Associated Noncoding RNA from the CCND1 Promoter. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_39
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DOI: https://doi.org/10.1007/978-1-61779-376-9_39
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