Abstract
It is now estimated that the human genome encodes thousands of long noncoding (lnc)RNAs. These novel molecules are causing a paradigm shift in the field of molecular biology as a number of lncRNAs have been shown to be involved in a wide range of biological functions including regulation of gene expression. Also, misregulation of lncRNAs has been observed in human diseases such as cancer and neurological disorders. These findings have spurred a huge interest in elucidating the functions and mechanisms of lncRNAs; and therefore, the need for new methods to do so. In this chapter, we discuss RIP-Seq, a method that is utilized to discover the lncRNA partners of a specific protein. This procedure involves immunoprecipitation of a protein from cross-linked cell lysate followed by reverse-cross-linking, isolation, and deep sequencing of RNAs, leading to the identification of all lncRNAs that are associated with a specific protein complex.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431:931–945
Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT, Stadler PF, Hertel J, Hackermuller J, Hofacker IL et al (2007) RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316:1484–1488
Wang J, Zhang J, Zheng H, Li J, Liu D, Li H, Samudrala R, Yu J, Wong GK (2004) Mouse transcriptome: neutral evolution of ‘non-coding’ complementary DNAs. Nature 431: 1 p following 757; discussion following 757
Nagano T, Fraser P (2011) No-nonsense functions for long noncoding RNAs. Cell 145:178–181
Guttman M, Donaghey J, Carey BW, Garber M, Grenier JK, Munson G, Young G, Lucas AB, Ach R, Bruhn L et al (2011) lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477:295–300
Clark MB, Mattick JS (2011) Long noncoding RNAs in cell biology. Semin Cell Dev Biol 22:366–376
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 25:1915–1927
Orom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q et al (2010) Long noncoding RNAs with enhancer-like function in human cells. Cell 143:46–58
Khalil A, Huarte M, Rinn J (2010) The emerging non-coding RNA World. In: Slack F (ed) MicroRNAs in development and cancer, vol 1. Imperial College Press, London, pp 17–44
Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A et al (2009) Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A 106:11667–11672
Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP et al (2009) Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458:223–227
Amaral PP, Dinger ME, Mercer TR, Mattick JS (2008) The eukaryotic genome as an RNA machine. Science 319:1787–1789
Carninci P, Hayashizaki Y (2007) Noncoding RNA transcription beyond annotated genes. Curr Opin Genet Dev 17:139–144
Ponting CP, Oliver PL, Reik W (2009) Evolution and functions of long noncoding RNAs. Cell 136:629–641
Lipovich L, Johnson R, Lin CY (2010) MacroRNA underdogs in a microRNA world: evolutionary, regulatory, and biomedical significance of mammalian long non-protein-coding RNA. Biochim Biophy 1799:597–615
Pontier DB, Gribnau J (2011) Xist regulation and function explored. Hum Genet 130:223–236
Augui S, Nora EP, Heard E (2011) Regulation of X-chromosome inactivation by the X-inactivation centre. Nat Rev Genet 12:429–442
Wapinski O, Chang HY (2011) Long noncoding RNAs and human disease. Trends Cell Biol 21:354–361
Khalil AM, Rinn JL (2011) RNA-protein interactions in human health and disease. Semin Cell Dev Biol 22(4):359–365
Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705
Khalil AM, Boyar FZ, Driscoll DJ (2004) Dynamic histone modifications mark sex chromosome inactivation and reactivation during mammalian spermatogenesis. Proc Natl Acad Sci U S A 101:16583–16587
Zhao J, Sun BK, Erwin JA, Song JJ, Lee JT (2008) Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322:750–756
Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E et al (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129:1311–1323
Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HY (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693
Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, Tsai MC, Hung T, Argani P, Rinn JL et al (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464:1071–1076
Sleutels F, Zwart R, Barlow DP (2002) The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415:810–813
Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC, Feil R, Fraser P (2008) The air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322:1717–1720
Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L, Komorowski J, Nagano T, Mancini-Dinardo D, Kanduri C (2008) Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell 32:232–246
Mohammad F, Mondal T, Guseva N, Pandey GK, Kanduri C (2010) Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1. Development 137:2493–2499
Meller VH, Gordadze PR, Park Y, Chu X, Stuckenholz C, Kelley RL, Kuroda MI (2000) Ordered assembly of roX RNAs into MSL complexes on the dosage-compensated X chromosome in Drosophila. Curr Biol 10:136–143
Kelley RL, Meller VH, Gordadze PR, Roman G, Davis RL, Kuroda MI (1999) Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98:513–522
Park Y, Kelley RL, Oh H, Kuroda MI, Meller VH (2002) Extent of chromatin spreading determined by roX RNA recruitment of MSL proteins. Science 298:1620–1623
Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, Tsai MJ, O’Malley BW (1999) A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97:17–27
Watanabe M, Yanagisawa J, Kitagawa H, Takeyama K, Ogawa S, Arao Y, Suzawa M, Kobayashi Y, Yano T, Yoshikawa H et al (2001) A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA. EMBO J 20:1341–1352
Kino T, Hurt DE, Ichijo T, Nader N, Chrousos GP (2010) Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor. Sci Signal 3:ra8
Niranjanakumari S, Lasda E, Brazas R, Garcia-Blanco MA (2002) Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26:182–190
Mili S, Steitz JA (2004) Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10:1692–1694
Zhao J, Ohsumi TK, Kung JT, Ogawa Y, Grau DJ, Sarma K, Song JJ, Kingston RE, Borowsky M, Lee JT (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40:939–953
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Moran, V.A., Niland, C.N., Khalil, A.M. (2012). Co-Immunoprecipitation of Long Noncoding RNAs. In: Engel, N. (eds) Genomic Imprinting. Methods in Molecular Biology, vol 925. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-011-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-011-3_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-010-6
Online ISBN: 978-1-62703-011-3
eBook Packages: Springer Protocols