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Isolation of Protein Complexes Associated with Long Noncoding RNAs

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Book cover Long Non-Coding RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1402))

Abstract

Long noncoding RNAs (lncRNAs) are a new class of regulatory genes that play critical roles in various processes ranging from normal development to human diseases. Recent studies have shown that protein complexes are required for the functions of lncRNAs. The identification of these proteins which are associated with lncRNAs is critical for the understanding of molecular mechanisms of lncRNAs in gene regulation and their functions. In this chapter, we describe a method to isolate proteins associated with lncRNAs. This procedure involves fusion protein maltose-binding protein (MBP) fused to MS2-binding protein to pull down the proteins associated with lncRNA and the identification of these proteins by mass spectrometry.

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References

  1. International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431:931–945

    Article  Google Scholar 

  2. Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT et al (2007) RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316:1484–1488

    Article  CAS  PubMed  Google Scholar 

  3. Guttman M, Donaghey J, Carey BW, Garber M, Grenier JK, Munson G et al (2011) lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477:295–300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Huarte M, Guttman M, Feldser D, Garber M, Koziol MJ, Kenzelmann-Broz D et al (2010) A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 142:409–419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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

    PubMed  PubMed Central  Google Scholar 

  6. Mancini-Dinardo D, Steele SJ, Levorse JM, Ingram RS, Tilghman SM (2006) Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes. Genes Dev 20:1268–1282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tian D, Sun S, Lee JT (2010) The long noncoding RNA, Jpx, is a molecular switch for X chromosome inactivation. Cell 143:390–403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Beltran M, Puig I, Pena C, Garcia JM, Alvarez AB, Pena R et al (2008) A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial-mesenchymal transition. Genes Dev 22:756–769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Benetatos L, Hatzimichael E, Dasoula A, Dranitsaris G, Tsiara S, Syrrou M et al (2010) CpG methylation analysis of the MEG3 and SNRPN imprinted genes in acute myeloid leukemia and myelodysplastic syndromes. Leuk Res 34:148–153

    Article  CAS  PubMed  Google Scholar 

  10. Calin GA, Liu CG, Ferracin M, Hyslop T, Spizzo R, Sevignani C et al (2007) Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell 12:215–229

    Article  CAS  PubMed  Google Scholar 

  11. Faghihi MA, Modarresi F, Khalil AM, Wood DE, Sahagan BG, Morgan TE et al (2008) Expression of a noncoding RNA is elevated in Alzheimer’s disease and drives rapid feed-forward regulation of beta-secretase. Nat Med 14:723–730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Harismendy O, Notani D, Song X, Rahim NG, Tanasa B, Heintzman N et al (2011) 9p21 DNA variants associated with coronary artery disease impair interferon-gamma signalling response. Nature 470:264–268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Li L, Feng T, Lian Y, Zhang G, Garen A, Song X (2009) Role of human noncoding RNAs in the control of tumorigenesis. Proc Natl Acad Sci U S A 106:12956–12961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mourtada-Maarabouni M, Pickard MR, Hedge VL, Farzaneh F, Williams GT (2009) GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer. Oncogene 28:195–208

    Article  CAS  PubMed  Google Scholar 

  15. Perez DS, Hoage TR, Pritchett JR, Ducharme-Smith AL, Halling ML, Ganapathiraju SC et al (2008) Long, abundantly expressed non-coding transcripts are altered in cancer. Hum Mol Genet 17:642–655

    Article  CAS  PubMed  Google Scholar 

  16. Petrovics G, Zhang W, Makarem M, Street JP, Connelly R, Sun L et al (2004) Elevated expression of PCGEM1, a prostate-specific gene with cell growth-promoting function, is associated with high-risk prostate cancer patients. Oncogene 23:605–611

    Article  CAS  PubMed  Google Scholar 

  17. Wang X, Arai S, Song X, Reichart D, Du K, Pascual G et al (2008) Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature 454:126–130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Azzalin CM, Reichenbach P, Khoriauli L, Giulotto E, Lingner J (2007) Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318:798–801

    Article  CAS  PubMed  Google Scholar 

  19. Bernard D, Prasanth KV, Tripathi V, Colasse S, Nakamura T, Xuan Z et al (2010) A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression. EMBO J 29:3082–3093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Flynn RL, Centore RC, O’Sullivan RJ, Rai R, Tse A, Songyang Z et al (2011) TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA. Nature 471:532–536

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gong C, Maquat LE (2011) lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements. Nature 470:284–288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gumireddy K, Li A, Yan J, Setoyama T, Johannes GJ, Orom UA et al (2013) Identification of a long non-coding RNA-associated RNP complex regulating metastasis at the translational step. EMBO J 32:2672–2684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Heo JB, Sung S (2011) Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA. Science 331:76–79

    Article  CAS  PubMed  Google Scholar 

  24. Kim TK, Hemberg M, Gray JM, Costa AM, Bear DM, Wu J et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465:182–187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M et al (2011) Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene. Oncogene 30:1956–1962

    Article  CAS  PubMed  Google Scholar 

  26. Liu F, Marquardt S, Lister C, Swiezewski S, Dean C (2010) Targeted 3′ processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing. Science 327:94–97

    Article  CAS  PubMed  Google Scholar 

  27. Maison C, Bailly D, Roche D, Montes de Oca R, Probst AV, Vassias I et al (2011) SUMOylation promotes de novo targeting of HP1alpha to pericentric heterochromatin. Nat Genet 43:220–227

    Article  CAS  PubMed  Google Scholar 

  28. Martianov I, Ramadass A, Serra Barros A, Chow N, Akoulitchev A (2007) Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript. Nature 445:666–670

    Article  CAS  PubMed  Google Scholar 

  29. Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC, Feil R et al (2008) The air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322:1717–1720

    Article  CAS  PubMed  Google Scholar 

  30. Orom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G et al (2010) Long noncoding RNAs with enhancer-like function in human cells. Cell 143:46–58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA et al (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129:1311–1323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Schmitz KM, Mayer C, Postepska A, Grummt I (2010) Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes. Genes Dev 24:2264–2269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT et al (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39:925–938

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F et al (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wang KC, Chang HY (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43:904–914

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yap KL, Li S, Munoz-Cabello AM, Raguz S, Zeng L, Mujtaba S et al (2010) Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell 38:662–674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgement

This work was supported by NIH/NCI grant R01CA148759, W. W. Smith Foundation, and Edward Mallinckrodt Jr. Foundation.

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Authors and Affiliations

  1. The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104, USA

    Kiranmai Gumireddy & Qihong Huang

  2. University of Washington Medical Center, 1959 N.E. Pacific Street, Seattle, WA, 98195, USA

    Jinchun Yan

Authors
  1. Kiranmai Gumireddy
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  2. Jinchun Yan
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  3. Qihong Huang
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Corresponding author

Correspondence to Qihong Huang .

Editor information

Editors and Affiliations

  1. School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Yi Feng

  2. School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Lin Zhang

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Gumireddy, K., Yan, J., Huang, Q. (2016). Isolation of Protein Complexes Associated with Long Noncoding RNAs. In: Feng, Y., Zhang, L. (eds) Long Non-Coding RNAs. Methods in Molecular Biology, vol 1402. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3378-5_4

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  • DOI: https://doi.org/10.1007/978-1-4939-3378-5_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3376-1

  • Online ISBN: 978-1-4939-3378-5

  • eBook Packages: Springer Protocols

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