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Genome-Wide Analysis of Long Noncoding RNA Turnover

  • Hidenori Tani
  • Naoto Imamachi
  • Rena Mizutani
  • Katsutoshi Imamura
  • Yeondae Kwon
  • Satoru Miyazaki
  • Sho Maekawa
  • Yutaka Suzuki
  • Nobuyoshi AkimitsuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1262)

Abstract

Genome-wide analysis for determining RNA turnover is an advanced method in RNA biology that examines the specific half-life of nuclear noncoding RNA (ncRNA). In particular, a pulse-labeling method using uridine analogs enables the determination of RNA stability under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5′-bromo-uridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing. The method is called BrU immunoprecipitation chase assay (BRIC) or BRIC through deep sequencing (BRIC-seq). Here, we describe a detailed protocol and technical tips for BRIC-seq.

Key words

Noncoding RNA RNA turnover RNA degradation 5′-bromo-uridine Deep sequencing BRIC-seq 

References

  1. 1.
    Schoenberg DR, Mquat LE (2012) Regulation of cytoplasmic mRNA decay. Nat Rev Genet 13:246–259PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Alonso CR (2012) A complex “mRNA degradation code” controls gene expression during animal development. Trends Genet 28:78–88PubMedCrossRefGoogle Scholar
  3. 3.
    Wilusz JE, Sunwoo H, Spector DL (2009) Long noncoding RNAs: functional surprises from the RNA world. Genes Dev 23:1494–1504PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Chen CY, Ezzeddine N, Shyu AB (2008) Messenger RNA half-life measurements in mammalian cells. Methods Enzymol 448:335–357PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Clark MB, Johnston RL, Inostroza-Ponta M, Fox AH, Fortini E, Moscato P, Dinger ME, Mattick JS (2012) Genome-wide analysis of long noncoding RNA stability. Genome Res 22:885–898PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Tani H, Mizutani R, Salam KA, Tano K, Ijiri K, Wakamatsu A, Isogai T, Suzuki Y, Akimitsu N (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived non-coding transcripts in mammals. Genome Res 22:947–956PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Sasaki YT, Ideue T, Sano M, Mituyama T, Hirose T (2009) MENepsilon/beta noncoding RNAs are essential for structural integrity of nuclear paraspeckles. Proc Natl Acad Sci U S A 106:2525–2530PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Tani H, Akimitsu N (2013) Genome-wide technology for determining RNA stability in mammalian cells: Historical perspective and recent advantages based on modified nucleotide labeling. RNA Biol 9:1233–1238CrossRefGoogle Scholar
  9. 9.
    Tani H, Imamachi N, Salam KA, Mizutani R, Ijiri K, Irie T, Yada T, Suzuki Y, Akimitsu N (2013) Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability. RNA Biol 9:1370–1379CrossRefGoogle Scholar
  10. 10.
    Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129:1311–1322PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A, Regev A, Lander ES, Rinn JL (2009) Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A 106:11667–11672PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Tani H, Torimura M (2013) Identification of short-lived long non-coding RNAs as surrogate indicators for chemical stress response. Biochem Biophys Res Commun 439:547–551PubMedCrossRefGoogle Scholar
  13. 13.
    Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Tani H, Torimura M, Akimitsu N (2013) The RNA degradation pathway regulates the function of GAS5 a non-coding RNA in mammalian cells. PLoS One 8:e55684PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Imamachi N, Tani H, Mizutani R, Imamura K, Irie T, Suzuki Y, Akimitsu N (2014) BRIC-seq: a genome-wide approach for determining RNA stability in mammalian cells. Methods 67:55–63PubMedCrossRefGoogle Scholar
  18. 18.
    Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, Rinn JL (2011) Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev 25:1915–1927PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, Guernec G, Martin D, Merkel A, Knowles DG, Lagarde J, Veeravalli L, Ruan X, Ruan Y, Lassmann T, Carninci P, Brown JB, Lipovich L, Gonzalez JM, Thomas M, Davis CA, Shiekhattar R, Gingeras TR, Hubbard TJ, Notredame C, Harrow J, Guigó R (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775–1789PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Xie C, Yuan J, Li H, Li M, Zhao G, Bu D, Zhu W, Wu W, Chen R, Zhao Y (2014) NONCODEv4: exploring the world of long non-coding RNA genes. Nucleic Acids Res 42(1):D98–D103PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Volders PJ, Helsens K, Wang X, Menten B, Martens L, Gevaert K, Vandesompele J, Mestdagh P (2013) LNCipedia: a database for annotated human lncRNA transcript sequences and structures. Nucleic acids Res 41:D246–D251PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Kim TK, Hemberg M, Gray JM, Costa AM, Bear DM, Wu J, Harmin DA, Laptewicz M, Barbara-Haley K, Kuersten S, Markenscoff-Papadimitriou E, Kuhl D, Bito H, Worley PF, Kreiman G, Greenberg ME (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465:182–187PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Ørom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R (2010) Long noncoding RNAs with enhancer-like function in human cells. Cell 143:46–58PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Hah N, Murakami S, Nagari A, Danko CG, Kraus WL (2013) Enhancer transcripts mark active estrogen receptor binding sites. Genome Res 23:1210–1223PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hidenori Tani
    • 1
  • Naoto Imamachi
    • 2
  • Rena Mizutani
    • 2
  • Katsutoshi Imamura
    • 2
  • Yeondae Kwon
    • 3
  • Satoru Miyazaki
    • 3
  • Sho Maekawa
    • 4
  • Yutaka Suzuki
    • 4
    • 5
  • Nobuyoshi Akimitsu
    • 2
    Email author
  1. 1.Research Institute for Environmental Management TechnologyNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  2. 2.Radioisotope CenterThe University of TokyoTokyoJapan
  3. 3.Department of Medical and Life Science, Faculty of Pharmaceutical ScienceTokyo University of ScienceNodaJapan
  4. 4.Department of Medical Genome Sciences, Graduate School of Frontier SciencesThe University of TokyoKashiwa-shiJapan
  5. 5.Department of Computational Biology, Graduate School of Frontier SciencesThe University of TokyoKashiwa-shiJapan

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