Analysis of the p53/CEP-1 regulated non-coding transcriptome in C. elegans by an NSR-seq strategy

Abstract

In recent years, large numbers of non-coding RNAs (ncRNAs) have been identified in C. elegans but their functions are still not well studied. In C. elegans, CEP-1 is the sole homolog of the p53 family of genes. In order to obtain transcription profiles of ncRNAs regulated by CEP-1 under normal and UV stressed conditions, we applied the ‘not-sorandom’ hexamers priming strategy to RNA sequencing in C. elegans, This NSR-seq strategy efficiently depleted rRNA transcripts from the samples and showed high technical replicability. We identified more than 1,000 ncRNAs whose apparent expression was repressed by CEP-1, while around 200 were activated. Around 40% of the CEP-1 activated ncRNAs promoters contain a putative CEP-1-binding site. CEP-1 regulated ncRNAs were frequently clustered and concentrated on the X chromosome. These results indicate that numerous ncRNAs are involved in CEP-1 transcriptional network and that these are especially enriched on the X chromosome in C. elegans.

Keywords

p53/CEP-1 / C. elegans / ncRNA / removal rRNA / NSR-seq / high technical replicability

References

[1] Adriaenssens E, Dumont L, Lottin S, Bolle D, Lepretre A, Delobelle A, Bouali F, Dugimont T, Coll J, Curgy JJ (1998) H19 overexpression in breast adenocarcinoma stromal cells is associated with tumor values and steroid receptor status but independent of p53 and Ki-67 expression. Am J Pathol153: 1597-1607

[2] Agostini M, Tucci P, Chen H, Knight RA, Bano D, Nicotera P, McKeon F, Melino G (2010) p73 regulates maintenance of neural stem cell. Biochem Biophys Res Commun403: 13-17

[3] Aird D, Ross MG, Chen WS, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A (2011) Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol12: R18

[4] Allen MA, Hillier LW, Waterston RH, Blumenthal T (2011) A global analysis of C. elegans trans-splicing. Genome Res21: 255-264

[5] Armour CD, Castle JC, Chen R, Babak T, Loerch P, Jackson S, Shah JK, Dey J, Rohl CA, Johnson JM (2009) Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat Methods6: 647-649

[6] Berkers CR, Maddocks OD, Cheung EC, Mor I, Vousden KH (2013) Metabolic regulation by p53 family members. Cell Metab18: 617-633

[7] Boominathan L (2010) The tumor suppressors p53, p63, and p73 are regulators of MicroRNA processing complex. PloS one5: e10615

[8] C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science282: 2012-2018

[9] Christov CP, Trivier E, Krude T (2008) Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation. Br J Cancer98: 981-988

[10] Crighton D, Woiwode A, Zhang C, Mandavia N, Morton JP, Warnock LJ, Milner J, White RJ, Johnson DL (2003) p53 represses RNA polymerase III transcription by targeting TBP and inhibiting promoter occupancy by TFIIIB. Embo J22: 2810-2820

[11] Deng W, Zhu XP, Skogerbo G, Zhao Y, Fu Z, Wang YD, He HS, Cai L, Sun H, Liu CN (2006) Organization of the Caenorhabditis elegans small non-coding transcriptome: Genomic features, biogenesis, and expression. Genome Res16: 20-29

[12] Derry WB, Putzke AP, Rothman JH (2001) Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance. Science294: 591-595

[13] Derry WB, Bierings R, van Iersel M, Satkunendran T, Reinke V, Rothman JH (2007) Regulation of developmental rate and germ cell proliferation in Caenorhabditis elegans by the p53 gene network. Cell Death Differ14: 662-670

[14] Dugimont T, Montpellier C, Adriaenssens E, Lottin S, Dumont L, Iotsova V, Lagrou C, Stehelin D, Coll J, Curgy JJ (1998) The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product p53. Oncogene16: 2395-2401

[15] Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F, Jacks T (2002) p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature416: 560-564

[16] Fong YY, Bender L, Wang WC, Strome S (2002) Regulation of the different chromatin states of autosomes and X chromosomes in the germ line of C. elegans. Science296: 2235-2238

[17] Green DR, Chipuk JE (2006) p53 and metabolism: inside the TIGAR. Cell126: 30-32

[18] Guttman M, Rinn JL (2012) Modular regulatory principles of large non-coding RNAs. Nature482: 339-346

[19] Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP (2009) Chromatin signature reveals over a thousand highly conserved large noncoding RNAs in mammals. Nature458: 223-227

[20] He H, Wang J, Liu T, Liu XS, Li T, Wang Y, Qian Z, Zheng H, Zhu X, Wu T (2007) Mapping the C. elegans noncoding transcriptome with a whole-genome tiling microarray. Genome Res17: 1471-1477

[21] Ho J, Benchimol S (2003) Transcriptional repression mediated by the p53 tumour suppressor. Cell Death Diff10: 404-408

[22] Huarte M, Rinn JL (2010) Large non-coding RNAs: missing links in cancer? Hum Mol Genet19: R152-R161

[23] Huarte M, Guttman M, Feldser D, Garber M, Koziol MJ, Kenzelmann-Broz D, Khalil AM, Zuk O, Amit I, Rabani M (2010) A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell142: 409-419

[24] Hung T, Wang YL, Lin MF, Koegel AK, Kotake Y, Grant GD, Horlings HM, Shah N, Umbricht C, Wang P (2011) Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters. Nat Genet43: 196-621

[25] Huyen Y, Jeffrey PD, Derry WB, Rothman JH, Pavletich NP, Stavridi ES, Halazonetis TD (2004) Structural differences in the DNA binding domains of human p53 and its C. elegans ortholog Cep-1. Structure12: 1237-1243

[26] Jiang L, Schlesinger F, Davis CA, Zhang Y, Li R, Salit M, Gingeras TR, Oliver B (2011) Synthetic spike-in standards for RNA-seq experiments. Genome Res21: 1543-1551

[27] Jones SJM, Riddle DL, Pouzyrev AT, Velculescu VE, Hillier L, Eddy SR, Stricklin SL, Baillie DL, Waterston R, Marra MA (2001) Changes in gene expression associated with developmental arrest and longevity in Caenorhabditis elegans. Genome Res11: 1346-1352

[28] Jones TA, Otto W, Marz M, Eddy SR, Stadler PF (2009) A survey of nematode SmY RNAs. RNA Biol6: 5-8

[29] Jung MS, Yun J, Chae HD, Kim JM, Kim SC, Choi TS, Shin DY (2001) p53 and its homologues, p63 and p73, induce a replicative senescence through inactivation of NF-Y transcription factor. Oncogene20: 5818-5825

[30] Kedde M, le Sage C, Duursma A, Zlotorynski E, van Leeuwen B, Nijkamp W, Beijersbergen R, Agami R (2006) Telomeraseindependent regulation of ATR by human telomerase RNA. J Biol Chem281: 40503-40514

[31] Kelly WG, Schaner CE, Dernburg AF, Lee MH, Kim SK, Villeneuve AM, Reinke V (2002) X-chromosome silencing in the germline of C. elegans. Development129: 479-492

[32] Kenzelmann Broz D, Spano Mello S, Bieging KT, Jiang D, Dusek RL, Brady CA, Sidow A, Attardi LD (2013) Global genomic profiling reveals an extensive p53-regulated autophagy program contributing to key p53 responses. Genes Dev27: 1016-1031

[33] Kirkpatrick KL, Mokbel K (2001) The significance of human telomerase reverse transcriptase (hTERT) in cancer. Eur J Surg Oncol27: 754-760

[34] Krastev DB, Slabicki M, Paszkowski-Rogacz M, Hubner NC, Junqueira M, Shevchenko A, Mann M, Neugebauer KM, Buchholz F (2011) A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly. Nat Cell Biol13: U186-U809

[35] Labbe JC, Hekimi S, Rokeach LA (1999) The levels of the RoRNPassociated Y RNA are dependent upon the presence of ROP-1, the Caenorhabditis elegans Ro60 protein. Genetics151: 143-150

[36] Labbe JC, Burgess J, Rokeach LA, Hekimi S (2000) ROP-1, an RNA quality-control pathway component, affects Caenorhabditis elegans dauer formation. Proc Natl Acad Sci USA97: 13233-13238

[37] Leonova KI, Brodsky L, Lipchick B, Pal M, Novototskaya L, Chenchik AA, Sen GC, Komarova EA, Gudkov AV (2013) p53 cooperates with DNA methylation and a suicidal interferon response to maintain epigenetic silencing of repeats and noncoding RNAs. Proc Natl Acad Sci USA110: E89-E98

[38] Levine AJ, Tomasini R, McKeon FD, Mak TW, Melino G (2011) The p53 family: guardians of maternal reproduction. Nat Rev Mol Cell Biol12: 259-265

[39] Li H, Cao Y, Berndt MC, Funder JW, Liu JP (1999) Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro. Oncogene18: 6785-6794

[40] Li A, Wei G, Wang Y, Zhou Y, Zhang XE, Bi L, Chen R (2012) Identification of intermediate-size non-coding RNAs involved in the UV-induced DNA damage response in C. elegans. PloS one7: e48066

[41] Loven J, Orlando DA, Sigova AA, Lin CY, Rahl PB, Burge CB, Levens DL, Lee TI, Young RA (2012) Revisiting global gene expression analysis. Cell151: 476-482

[42] Lowe J, Shatz M, Resnick M, Menendez D (2013) Modulation of immune responses by the tumor suppressor p53. BioDiscovery8: 2

[43] Lu ZJ, Yip KY, Wang G, Shou C, Hillier LW, Khurana E, Agarwal A, Auerbach R, Rozowsky J, Cheng C (2011) Prediction and characterization of noncoding RNAs in C. elegans by integrating conservation, secondary structure, and high-throughput sequencing and array data. Genome Res21: 276-285

[44] Lu KH, Li W, Liu XH, Sun M, Zhang ML, Wu WQ, Xie WP, Hou YY (2013) Long non-coding RNA MEG3 inhibits NSCLC cells proliferation and induces apoptosis by affecting p53 expression. BMC Cancer13: 461

[45] MacMorris M, Kumar M, Lasda E, Larsen A, Kraemer B, Blumenthal T (2007) A novel family of C. elegans snRNPs contains proteins associated with trans-splicing. RNA-A Publ RNA Soc13: 511-520

[46] Maiuri MC, Galluzzi L, Morselli E, Kepp O, Malik SA, Kroemer G (2010) Autophagy regulation by p53. Curr Opin Cell Biol22: 181-185

[47] Maroney PA, Yu YT, Jankowska M, Nilsen TW (1996) Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs. RNA-A Publ RNA Soc2: 735-745

[48] Matouk IJ, Mezan S, Mizrahi A, Ohana P, Abu-lail R, Fellig Y, deGroot N, Galun E, Hochberg A (2010) The oncofetal H19 RNA connection: hypoxia, p53 and cancer. BBA-Mol Cell Res1803: 443-451

[49] Melino G, Lu X, Gasco M, Crook T, Knight RA (2003) Functional regulation of p73 and p63: development and cancer. Trends Biochem Sci28: 663-670

[50] Menendez D, Inga A, Resnick MA (2009) The expanding universe of p53 targets. Nat Rev Cancer9: 724-737

[51] Molchadsky A, Shats I, Goldfinger N, Pevsner-Fischer M, Olson M, Rinon A, Tzahor E, Lozano G, Zipori D, Sarig R (2008) p53 plays a role in mesenchymal differentiation programs, in a cell fate dependent manner. PloS One3: e3707

[52] Mondal AM, Horikawa I, Pine SR, Fujita K, Morgan KM, Vera E, Mazur SJ, Appella E, Vojtesek B, Blasco MA (2013) p53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes. J Clin Investig123: 5247-5257

[53] Nam JW, Bartel DP (2012) Long noncoding RNAs in C. elegans. Genome Res22: 2529-2540

[54] Ou HD, Lohr F, Vogel V, Mantele W, Dotsch V (2007) Structural evolution of C-terminal domains in the p53 family. EMBO J26: 3463-3473

[55] Riley T, Sontag E, Chen P, Levine A (2008) Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol9: 402-412

[56] Rinn JL, Loewer S, Huarte M, Cabili M, Guttman M, Regev A, Lander ES, Daley GQ, Rinn JL (2011) Large intergenic non-coding RNAs in chromatin, cancer and stem cells. FASEB J25

[57] Roberts A, Trapnell C, Donaghey J, Rinn JL, Pachter L (2011) Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol12: R22

[58] Roger L, Gadea G, Roux P (2006) Control of cell migration: a tumour suppressor function for p53? Biol Cell Under Auspices Eur Cell Biol Organ98: 141-152

[59] Sabapathy K, Klemm M, Jaenisch R, Wagner EF (1997) Regulation of ES cell differentiation by functional and conformational modulation of p53. EMBO J16: 6217-6229

[60] Schumacher B, Hofmann K, Boulton S, Gartner A (2001) The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis. Curr Biol11: 1722-1727

[61] Senoo M, Pinto F, Crum CP, McKeon F (2007) p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell129: 523-536

[62] Stergiou L, Doukoumetzidis K, Sendoel A, Hengartner MO (2007) The nucleotide excision repair pathway is required for UV-Cinduced apoptosis in Caenorhabditis elegans. Cell Death Diff14: 1129-1138

[63] Stiewe T, Zimmermann S, Frilling A, Esche H, Putzer BM (2002) Transactivation-deficient Delta TA-p73 acts as an oncogene. Cancer Res62: 3598-3602

[64] Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K (2009) Modulation of microRNA processing by p53. Nature460: 529-533

[65] Venter JC, Adams MD, Myers EW (2001) The sequence of the human genome. Science291: 1304-1351

[66] Wang Y, Chen J, Wei G, He H, Zhu X, Xiao T, Yuan J, Dong B, He S, Skogerbo G (2011) The Caenorhabditis elegans intermediate- size transcriptome shows high degree of stage-specific expression. Nucleic Acids Res39: 5203-5214

[67] Xiao T, Wang Y, Luo H, Liu L, Wei G, Chen X, Sun Y, Chen X, Skogerbo G, Chen R (2012) A differential sequencing-based analysis of the C. elegans noncoding transcriptome. RNA18: 626-639

[68] Xu DW, Wang Q, Gruber A, Bjorkholm M, Chen ZG, Zaid A, Selivanova G, Peterson C, Wiman KG, Pisa P (2000) Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells. Oncogene19: 5123-5133

[69] Yang A, McKeon F (2000) P63 and P73: P53 mimics, menaces and more. Nat Rev Mol Cell Biol1: 199-207

[70] Yang A, Zhu Z, Kettenbach A, Kapranov P, McKeon F, Gingeras TRStruhl K (2010) Genome-wide mapping indicates that p73 and p63 co-occupy target sites and have similar dna-binding profiles in vivo. PloS One5: e11572

[71] Yang F, Bi J, Xue X, Zheng L, Zhi K, Hua J, Fang G (2012) Upregulated long non-coding RNA H19 contributes to proliferation of gastric cancer cells. FEBS J279: 3159-3165

[72] Yu X, Harris SL, Levine AJ (2006) The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res66: 4795-4801

[73] Zhang L, Yu D, Hu M, Xiong S, Lang A, Ellis LM, Pollock RE (2000) Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Cancer Res60: 3655-3661

[74] Zhou Y, Zhong Y, Wang Y, Zhang X, Batista DL, Gejman R, Ansell PJ, Zhao J, Weng C, Klibanski A (2007) Activation of p53 by MEG3 non-coding RNA. J Biol Chem282: 24731-24742

RIGHTS & PERMISSIONS

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.