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Promoter-Associated Noncoding RNA from the CCND1 Promoter

  • Xiaoyuan Song
  • Xiangting Wang
  • Shigeki Arai
  • Riki KurokawaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 809)

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.

Key words

Promoter-associated noncoding RNA Translocated in liposarcoma RNA immunoprecipitation Chromatin immunoprecipitation Gel-shift assay RNA oligonucleotide 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Xiaoyuan Song
    • 1
  • Xiangting Wang
    • 1
  • Shigeki Arai
    • 2
  • Riki Kurokawa
    • 2
    Email author
  1. 1.Department of MedicineUniversity of California, San Diego School of MedicineLa JollaUSA
  2. 2.Division of Gene Structure and FunctionResearch Center for Genomic Medicine, Saitama Medical UniversitySaitama-KenJapan

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