Abstract
A detailed understanding of post-transcriptional gene expression is necessary to correlate the different elements involved in the many levels of RNA–protein interactions that are needed to coordinate the cellular biomolecular machinery. The profile of mRNA, a major component of this machinery, can be examined after isolation from specific RNA-binding proteins (RBPs). RIP-Chip or ribonomic profiling is a versatilein vivo technique that has been widely used to study post-transcriptional gene regulation and the localization of mRNA. Here we elaborately detail the methodology for mRNA isolation using RBP immunoprecipitation (RIP) as a primary approach. Specific antibodies are used to target RBPs, which are then used to capture the associated mRNA.
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Futcher, B., Latter, G. I., Monardo, P., McLaughlin, C. S., and Garrels, J. I. (1999) A sampling of the yeast proteome. Mol Cell Biol. 19, 7357–7368.
Gygi, S. P., Rochon, Y., Franza, B. R., and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 19, 1720–1730.
Tenenbaum, S. A., Carson, C. C., Lager, P. J., and Keene, J. D. (2000) Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A. 97, 14085–14090.
Perou, C. M., Jeffrey, S. S., van de Rijn, M., Rees, C. A., Eisen, M. B., Ross, D. T., Pergamenschikov, A., Williams, C. F., Zhu, S. X., Lee, J. C., Lashkari, D., Shalon, D., Brown, P. O., and Botstein, D. (1999) Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A. 96, 9212–9217.
Townley-Tilson, W. H., Pendergrass, S. A., Marzluff, W. F., and Whitfield, M. L. (2006) Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein. RNA. 12, 1853–1867.
Sanchez-Diaz, P., and Penalva, L. O. (2006) Post-transcription meets post-genomic: the saga of RNA binding proteins in a new era. RNA Biol. 3, 101–109.
Lockhart, D. J., and Winzeler, E. A. (2000) Genomics, gene expression and DNA arrays. Nature. 405, 827–836.
Mata, J., Marguerat, S., and Bahler, J. (2005) Post-transcriptional control of gene expression: a genome-wide perspective. Trends Biochem Sci. 30, 506–514.
Tenenbaum, S. A., Lager, P. J., Carson, C. C., and Keene, J. D. (2002) Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. Methods. 26, 191–198.
Keene, J. D., Komisarow, J. M., and Friedersdorf, M. B. (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc. 1, 302–307.
Brown, V., Jin, P., Ceman, S., Darnell, J. C., O’Donnell, W. T., Tenenbaum, S. A., Jin, X., Feng, Y., Wilkinson, K. D., Keene, J. D., Darnell, R. B., and Warren, S. T. (2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell. 107, 477–487.
Darnell, J. C., Jensen, K. B., Jin, P., Brown, V., Warren, S. T., and Darnell, R. B. (2001) Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell. 107, 489–499.
Wang, W. X., Wilfred, B. R., Hu, Y., Stromberg, A. J., and Nelson, P. T. (2010) Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes. RNA. 16, 394–404.
Gerber, A. P., Herschlag, D., and Brown, P. O. (2004) Extensive Association of Functionally and Cytotopically Related mRNAs with Puf Family RNA-Binding Proteins in Yeast. PLoS Biol. 2, E79.
Lopez de Silanes, I., Zhan, M., Lal, A., Yang, X., and Gorospe, M. (2004) Identification of a target RNA motif for RNA-binding protein HuR. Proc Natl Acad Sci U S A. 101, 2987–2992.
Mazan-Mamczarz, K., Hagner, P. R., Corl, S., Srikantan, S., Wood, W. H., Becker, K. G., Gorospe, M., Keene, J. D., Levenson, A. S., and Gartenhaus, R. B. (2008) Post-transcriptional gene regulation by HuR promotes a more tumorigenic phenotype. Oncogene. 27, 6151–6163.
Baroni, T. E., Chittur, S. V., George, A. D., and Tenenbaum, S. A. (2008) Advances in RIP-chip analysis : RNA-binding protein immunoprecipitation-microarray profiling. Methods Mol Biol. 419, 93–108.
Gyorffy, B., Molnar, B., Lage, H., Szallasi, Z., and Eklund, A. C. (2009) Evaluation of microarray preprocessing algorithms based on concordance with RT-PCR in clinical samples. PLoS One. 4, e5645.
Seo, J., and Hoffman, E. P. (2006) Probe set algorithms: is there a rational best bet?. BMC Bioinformatics. 7, 395.
Benjamini, Y., and Hochberg, Y. (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. Roy. Statist. Soc. Ser. 57, 289–300.
Birney, E., Stamatoyannopoulos, J. A., Dutta, A., Guigo, R., Gingeras, T. R., Margulies, E. H., Weng, Z., Snyder, M., Dermitzakis, E. T., Thurman, R. E., Kuehn, M. S., Taylor, C. M., Neph, S., Koch, C. M., Asthana, S., Malhotra, A., Adzhubei, I., Greenbaum, J. A., Andrews, R. M., Flicek, P., Boyle, P. J., Cao, H., Carter, N. P., Clelland, G. K., Davis, S., Day, N., Dhami, P., Dillon, S. C., Dorschner, M. O., Fiegler, H., Giresi, P. G., Goldy, J., Hawrylycz, M., Haydock, A., Humbert, R., James, K. D., Johnson, B. E., Johnson, E. M., Frum, T. T., Rosenzweig, E. R., Karnani, N., Lee, K., Lefebvre, G. C., Navas, P. A., Neri, F., Parker, S. C., Sabo, P. J., Sandstrom, R., Shafer, A., Vetrie, D., Weaver, M., Wilcox, S., Yu, M., Collins, F. S., Dekker, J., Lieb, J. D., Tullius, T. D., Crawford, G. E., Sunyaev, S., Noble, W. S., Dunham, I., Denoeud, F., Reymond, A., Kapranov, P., Rozowsky, J., Zheng, D., Castelo, R., Frankish, A., Harrow, J., Ghosh, S., Sandelin, A., Hofacker, I. L., Baertsch, R., Keefe, D., Dike, S., Cheng, J., Hirsch, H. A., Sekinger, E. A., Lagarde, J., Abril, J. F., Shahab, A., Flamm, C., Fried, C., Hackermuller, J., Hertel, J., Lindemeyer, M., Missal, K., Tanzer, A., Washietl, S., Korbel, J., Emanuelsson, O., Pedersen, J. S., Holroyd, N., Taylor, R., Swarbreck, D., Matthews, N., Dickson, M. C., Thomas, D. J., Weirauch, M. T., Gilbert, J., Drenkow, J., Bell, I., Zhao, X., Srinivasan, K. G., Sung, W. K., Ooi, H. S., Chiu, K. P., Foissac, S., Alioto, T., Brent, M., Pachter, L., Tress, M. L., Valencia, A., Choo, S. W., Choo, C. Y., Ucla, C., Manzano, C., Wyss, C., Cheung, E., Clark, T. G., Brown, J. B., Ganesh, M., Patel, S., Tammana, H., Chrast, J., Henrichsen, C. N., Kai, C., Kawai, J., Nagalakshmi, U., Wu, J., Lian, Z., Lian, J., Newburger, P., Zhang, X., Bickel, P., Mattick, J. S., Carninci, P., Hayashizaki, Y., Weissman, S., Hubbard, T., Myers, R. M., Rogers, J., Stadler, P. F., Lowe, T. M., Wei, C. L., Ruan, Y., Struhl, K., Gerstein, M., Antonarakis, S. E., Fu, Y., Green, E. D., Karaoz, U., Siepel, A., Taylor, J., Liefer, L. A., Wetterstrand, K. A., Good, P. J., Feingold, E. A., Guyer, M. S., Cooper, G. M., Asimenos, G., Dewey, C. N., Hou, M., Nikolaev, S., Montoya-Burgos, J. I., Loytynoja, A., Whelan, S., Pardi, F., Massingham, T., Huang, H., Zhang, N. R., Holmes, I., Mullikin, J. C., Ureta-Vidal, A., Paten, B., Seringhaus, M., Church, D., Rosenbloom, K., Kent, W. J., Stone, E. A., Batzoglou, S., Goldman, N., Hardison, R. C., Haussler, D., Miller, W., Sidow, A., Trinklein, N. D., Zhang, Z. D., Barrera, L., Stuart, R., King, D. C., Ameur, A., Enroth, S., Bieda, M. C., Kim, J., Bhinge, A. A., Jiang, N., Liu, J., Yao, F., Vega, V. B., Lee, C. W., Ng, P., Yang, A., Moqtaderi, Z., Zhu, Z., Xu, X., Squazzo, S., Oberley, M. J., Inman, D., Singer, M. A., Richmond, T. A., Munn, K. J., Rada-Iglesias, A., Wallerman, O., Komorowski, J., Fowler, J. C., Couttet, P., Bruce, A. W., Dovey, O. M., Ellis, P. D., Langford, C. F., Nix, D. A., Euskirchen, G., Hartman, S., Urban, A. E., Kraus, P., Van Calcar, S., Heintzman, N., Kim, T. H., Wang, K., Qu, C., Hon, G., Luna, R., Glass, C. K., Rosenfeld, M. G., Aldred, S. F., Cooper, S. J., Halees, A., Lin, J. M., Shulha, H. P., Xu, M., Haidar, J. N., Yu, Y., Iyer, V. R., Green, R. D., Wadelius, C., Farnham, P. J., Ren, B., Harte, R. A., Hinrichs, A. S., Trumbower, H., Clawson, H., Hillman-Jackson, J., Zweig, A. S., Smith, K., Thakkapallayil, A., Barber, G., Kuhn, R. M., Karolchik, D., Armengol, L., Bird, C. P., de Bakker, P. I., Kern, A. D., Lopez-Bigas, N., Martin, J. D., Stranger, B. E., Woodroffe, A., Davydov, E., Dimas, A., Eyras, E., Hallgrimsdottir, I. B., Huppert, J., Zody, M. C., Abecasis, G. R., Estivill, X., Bouffard, G. G., Guan, X., Hansen, N. F., Idol, J. R., Maduro, V. V., Maskeri, B., McDowell, J. C., Park, M., Thomas, P. J., Young, A. C., Blakesley, R. W., Muzny, D. M., Sodergren, E., Wheeler, D. A., Worley, K. C., Jiang, H., Weinstock, G. M., Gibbs, R. A., Graves, T., Fulton, R., Mardis, E. R., Wilson, R. K., Clamp, M., Cuff, J., Gnerre, S., Jaffe, D. B., Chang, J. L., Lindblad-Toh, K., Lander, E. S., Koriabine, M., Nefedov, M., Osoegawa, K., Yoshinaga, Y., Zhu, B., and de Jong, P. J. (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 447, 799–816.
Kurien, B. T., and Scofield, R. H. (2006) Western blotting. Methods. 38, 283–293.
Lasne, F. (2001) Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures. J Immunol Methods. 253, 125–131.
Macphee, D. J. (2009) Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods.
Zhang, X., Zou, T., Rao, J. N., Liu, L., Xiao, L., Wang, P. Y., Cui, Y. H., Gorospe, M., and Wang, J. Y. (2009) Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines. Nucleic Acids Res. 37, 7623–7637.
Lisbin, M. J., Qiu, J., and White, K. (2001) The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA. Genes Dev. 15, 2546–2561.
Acknowledgments
We wish to thank the members of the Tenenbaum Lab and specifically Arthur Beauregard for helpful editorial comments. This work was supported in part by NIH/NHGRI Grant 5U01HG004571 to SAT.
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Jayaseelan, S., Doyle, F., Currenti, S., Tenenbaum, S.A. (2011). RIP: An mRNA Localization Technique. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_25
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DOI: https://doi.org/10.1007/978-1-61779-005-8_25
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