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RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts

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Abstract

RNA targets of multitargeted RNA-binding proteins (RBPs) can be studied by various methods including mobility shift assays, iterative in vitro selection techniques and computational approaches. These techniques, however, cannot be used to identify the cellular context within which mRNAs associate, nor can they be used to elucidate the dynamic composition of RNAs in ribonucleoprotein (RNP) complexes in response to physiological stimuli. But by combining biochemical and genomics procedures to isolate and identify RNAs associated with RNA-binding proteins, information regarding RNA–protein and RNA–RNA interactions can be examined more directly within a cellular context. Several protocols — including the yeast three-hybrid system and immunoprecipitations that use physical or chemical cross-linking — have been developed to address this issue. Cross-linking procedures in general, however, are limited by inefficiency and sequence biases. The approach outlined here, termed RNP immunoprecipitation–microarray (RIP-Chip), allows the identification of discrete subsets of RNAs associated with multi-targeted RNA-binding proteins and provides information regarding changes in the intracellular composition of mRNPs in response to physical, chemical or developmental inducements of living systems. Thus, RIP-Chip can be used to identify subsets of RNAs that have related functions and are potentially co-regulated, as well as proteins that are associated with them in RNP complexes. Using RIP-Chip, the identification and/or quantification of RNAs in RNP complexes can be accomplished within a few hours or days depending on the RNA detection method used.

*Note: In the version of the article originally published, in the last sentence of the ANTICIPATED RESULTS section, the callout should be to reference 19 instead of 18. The error has been corrected in the HTML and PDF versions of the article.

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  • 31 August 2006

    In the version of the article originally published, in the last sentence of the ANTICIPATED RESULTS section, the callout should be to reference 19 instead of 18. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Ideker, T. et al. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 292, 929–934 (2001).

    CAS  PubMed  Google Scholar 

  2. Griffin, T.J. et al. Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae. Mol. Cell. Proteomics 1, 323–333 (2002).

    CAS  PubMed  Google Scholar 

  3. Keene, J.D. Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome. Proc. Natl. Acad. Sci. USA 98, 7018–7024 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Keene, J.D. & Tenenbaum, S.A. Eukaryotic mRNPs may represent posttranscriptional operons. Mol. Cell 9, 1161–1167 (2002).

    CAS  PubMed  Google Scholar 

  5. Hieronymus, H. & Silver, P.A. A systems view of mRNP biology. Genes Dev. 18, 2845–2860 (2004).

    CAS  PubMed  Google Scholar 

  6. Keene, J.D. & Lager, P.J. Posttranscriptional operons and regulons coordinating gene expression. Chr. Res. 13, 327–337 (2005).

    CAS  Google Scholar 

  7. Moore, M.J. From birth to death: the complex lives of eukaryotic mRNAs. Science 309, 1514–1518 (2005).

    CAS  PubMed  Google Scholar 

  8. Cilley, C.D. & Williamson, J.R. PACE Analysis of RNA-peptide interactions. In Methods in Molecular Biology (S.R. Haynes, ed.) 129–142 (Humana Press, Totowa, New Jersey, 1999)

    Google Scholar 

  9. Gao, F., Carson, C., Levine, T.D. & Keene, J.D. Selection of a subset of mRNAs from 3′UTR combinatorial libraries using neuronal RNA-binding protein, Hel-N1. Proc. Natl. Acad. Sci. USA 91, 11207–11211 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. SenGupta, D.J. et al. A three-hybrid system to detect RNA-protein interactions in vivo. Proc. Natl. Acad. Sci. USA 93, 8496–8501 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bernstein, D.S., Buter, N., Stumpf, C. & Wickens, M. Analyzing mRNA-protein complexes using a yeast three-hybrid system. Methods 26, 123–141 (2002).

    CAS  PubMed  Google Scholar 

  12. Wang, Z.F. et al. The protein that binds the 3′ end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing. Genes Dev. 10, 3028–3040 (1996).

    CAS  PubMed  Google Scholar 

  13. Tenenbaum, S.A., Carson, C.C., Lager, P.J. & Keene, J.D. Identifying mRNA subsets in mRNP complexes using cDNA arrays. Proc. Natl. Acad. Sci. USA 97, 14085–14090 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Tenenbaum, S.A., Lager, P.J., Carson, C.C. & Keene, J.D. Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. Methods 26, 191–198 (2002).

    CAS  PubMed  Google Scholar 

  15. Tenenbaum, S.A., Carson, C.C., Atasoy, U. & Keene, J.D. Genome-wide regulatory analysis combining en masse nuclear run-ons (emRUNs) and ribonomic profiling. Gene 317, 79–87 (2003).

    CAS  PubMed  Google Scholar 

  16. Eystathioy, T. et al. A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Mol. Biol. Cell 13, 1338–1351 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Kaneko, S. & Manley, J.L. The mammalian RNA polymerase II C-terminal domain interacts with RNA to suppress transcription-coupled 3′ end formation. Mol. Cell 20, 91–103 (2005).

    CAS  PubMed  Google Scholar 

  18. Mili, S. & Steitz, J.A. Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10, 1692–1694 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Penalva, L.O.F. Burdick, M.D., Lin, S.M., Sutterluety, H. & Keene, J.D. RNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells. Mol. Can. 3, 24–35 (2004).

    Google Scholar 

  20. Penalva, O.F., Tenenbaum, S.A. & Keene, J.D. Gene expression analysis of messenger RNP complexes. Meth. Mol. Biol. 257, 125–134 (2004).

    CAS  Google Scholar 

  21. Roy, P.J., Stuart, J.M., Lund, J. & Kim, S.K. Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature 418, 975–979 (2002).

    CAS  PubMed  Google Scholar 

  22. Niranjanakumari, S., Lasda, E., Brazas, R. & Garcia-Blanco, M.A. Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26, 182–190 (2002).

    CAS  PubMed  Google Scholar 

  23. Ule, J., Jensen, K., Mele, A. & Darnell, R.B. CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37, 376–386 (2005).

    CAS  PubMed  Google Scholar 

  24. Zang, Z., Edenberg, H.J. & Davis, R.L. Isolation of mRNA from specific tissues of Drosophilia by mRNA tagging. Nuc. Acids Res. 33, e148 (2005).

    Google Scholar 

  25. Kunitomo, H., Uesugi, H., Kohara, Y. & Iino, Y. Identification of ciliated sensory neuron-expressed genes in Caenorhabditis elegans using targeted pull-down of poly(A) tails. Genome Biol. 6, R17 (2005).

    Google Scholar 

  26. Ule, J. et al. CLIP identifies Nova-regulated RNA networks in the brain. Science 302, 1212–1215 (2003).

    CAS  PubMed  Google Scholar 

  27. Gerber, A.P., Herschlag, D. & Brown, P.O. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol. 2, E79 (2004).

    PubMed  PubMed Central  Google Scholar 

  28. Gerber, A.P., Luschnig, S., Kransow, M.A., Brown, P.O. & Herschlag, D. Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 103, 4487–4492 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Penalva, P.O. & Keene, J.D. Biotinylated tags for recovery and characterization of ribonucleoprotein complexes. Biotechniques 37, 604–610 (2004).

    CAS  PubMed  Google Scholar 

  30. Antic, D., Lu, N. & Keene, J.D. ELAV tumor antigen, Hel-N1, increases translation of neurofilament M mRNA and induces formation of neurites in human teratocarcinoma cells. Genes Dev. 13, 449–461 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhu, J., Shendure, J., Mitra, R.D. & Church, G.M. Single molecule profiling of alternative pre-mRNA splicing. Science 301, 836–838 (2003).

    CAS  PubMed  Google Scholar 

  32. Kapranov, P. et al. Large-scale transcriptional activity in chromosomes 21 and 22. Science 296, 916–919 (2002).

    CAS  PubMed  Google Scholar 

  33. Thomson, J.M., Parker, J., Perou, C.M. & Hammond, S.M. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods. 1, 47–53 (2004).

    CAS  PubMed  Google Scholar 

  34. Zhang, B., Kirov, S. & Snoody, J. WebGestalt: and integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res. 33, W741–748 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Brown, V. et al. Microarray identification of Fragile X-Mental Retardation protein (FMRP)-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107, 477–487 (2001).

    CAS  PubMed  Google Scholar 

  36. Lopez de Silanes, I., Zhan, M., Lal, A., Yang, X. & Gorospe, M. Identification of a target RNA motif for RNA-binding protein HuR. Proc. Natl. Acad. Sci. USA 101, 2987–2992 (2004).

    PubMed  Google Scholar 

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Correspondence to Jack D Keene, Jordan M Komisarow or Matthew B Friedersdorf.

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J.D.K. holds stock in Ribonomics, Inc., a company that owns patents for the RIP-Chip technology.

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Keene, J., Komisarow, J. & Friedersdorf, M. RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc 1, 302–307 (2006). https://doi.org/10.1038/nprot.2006.47

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