Abstract
RNA-based regulation is increasingly recognized as an important factor shaping the cellular transcriptome. RNA-binding proteins that interact with cis-regulatory motifs within pre-mRNAs determine the fate of their targets. Understanding posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe RNA immunoprecipitation in Arabidopsis thaliana. Transgenic plants expressing an RNA-binding protein fused to green fluorescent protein are treated with formaldehyde to “trap” RNAs in complexes with their physiological protein partners. A whole-cell extract is subjected to immunoprecipitation with an antibody against the GFP tag. In parallel, a mock immunoprecipitation is performed using an unrelated antibody. Coprecipitated RNAs are eluted from the immunoprecipitate and identified via real-time PCR. Enrichment relative to immunoprecipitation from plants expressing GFP only and mock immunoprecipitation with an unrelated antibody indicates specific binding.
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References
Keene JD, Tenenbaum SA (2002) Eukaryotic mRNPs may represent posttranscriptional operons. Mol Cell 9:1161–1167
Tenenbaum SA, Carson CC, Lager PJ et al (2000) Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A 97:14085–14090
Cheng Y, Chen X (2004) Posttranscriptional control of plant development. Curr Opin Plant Biol 7:20–25
Lorkovic ZJ (2009) Role of plant RNA-binding proteins in development, stress response and genome organization. Trends Plant Sci 14:229–236
Staiger D, Green R (2011) RNA-based regulation in the plant circadian clock. Trends Plant Sci 16:517–523
Quesada V, Dean C, Simpson GG (2005) Regulated RNA processing in the control of Arabidopsis flowering. Int J Dev Biol 49:773–780
Gorlach M, Burd CG, Dreyfuss G (1994) The determinants of RNA-binding specificity of the heterogeneous nuclear ribonucleoprotein C proteins. J Biol Chem 269:23074–23078
Lorenz C, von Pelchrzim F, Schroeder R (2006) Genomic systematic evolution of ligands by exponential enrichment (Genomic SELEX) for the identification of protein-binding RNAs independent of their expression levels. Nat Protoc 1:2204–2212
Brown JW, Birmingham A, Griffiths PE et al (2009) The RNA structure alignment ontology. RNA 15:1623–1631
Kaufmann K, Muino JM, Osteras M et al (2010) Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat Protoc 5:457–472
Schöning JC, Streitner C, Page DR et al (2007) Autoregulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation. Plant J 52:1119–1130
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
Terzi LC, Simpson GG (2009) Arabidopsis RNA immunoprecipitation. Plant J 59:163–168
Ule J, Jensen KB, Ruggiu M et al (2003) CLIP identifies Nova-regulated RNA networks in the brain. Science 302:1212–1215
Hafner M, Landthaler M, Burger L et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141
Mukherjee N, Corcoran DL, Nusbaum JD et al (2011) Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability. Mol Cell 43:327–339
Frohnmeyer H, Staiger D (2003) Ultraviolet-B radiation-mediated responses in plants. Balancing damage and protection. Plant Physiol 133:1420–1428
Rizzini L, Favory JJ, Cloix C et al (2011) Perception of UV-B by the Arabidopsis UVR8 protein. Science 332:103–106
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Haring M, Offermann S, Danker T et al (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3:11
Galgano A, Gerber AP (2011) RNA-binding protein immunopurification-microarray (RIP-Chip) analysis to profile localized RNAs. Methods Mol Biol 714:369–385
Jensen KB, Darnell RB (2008) CLIP: crosslinking and immunoprecipitation of in vivo RNA targets of RNA-binding proteins. Methods Mol Biol 488:85–98
Schmitz-Linneweber C, Williams-Carrier R, Barkan A (2005) RNA immunoprecipitation and microarray analysis show a chloroplast Pentatricopeptide repeat protein to be associated with the 5′ region of mRNAs whose translation it activates. Plant Cell 17:2791–2804
Barkan A (2009) Genome-wide analysis of RNA-protein interactions in plants. Methods Mol Biol 553:13–37
Montgomery TA, Howell MD, Cuperus JT et al (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133:128–141
Mustroph A, Juntawong P, Bailey-Serres J (2009) Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods. Methods Mol Biol 553:109–126
Staiger D, Heintzen C (1999) The circadian system of Arabidopsis thaliana: forward and reverse genetic approaches. Chronobiol Int 16:1–16
Staiger D, Zecca L, Wieczorek Kirk DA et al (2003) The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA. Plant J 33:361–371
Schöning JC, Streitner C, Meyer IM et al (2008) Reciprocal regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop coupling alternative splicing to nonsense-mediated decay in Arabidopsis. Nucleic Acids Res 36:6977–6987
Streitner C, Hennig L, Korneli C et al (2010) Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7. BMC Plant Biol 10:221
Rothbauer U, Zolghadr K, Tillib S et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3:887–889
Rothbauer U, Zolghadr K, Muyldermans S et al (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7:282–289
Isono E, Schwechheimer C (2010) Co-immunoprecipitation and protein blots. Methods Mol Biol 655:377–387
Trinkle-Mulcahy L, Boulon S, Lam YW et al (2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol 183:223–239
Niranjanakumari S, Lasda E, Brazas R et al (2002) Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26:182–190
Hummon AB, Lim SR, Difilippantonio MJ et al (2007) Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage. Biotechniques 42:467–472
Acknowledgement
We thank Dr. Gordon Simpson (Dundee) for discussions on RIP strategies.
This work was supported by the DFG (STA 653/2 and SFB 613). T.K. is a fellow of the German National Academic Foundation.
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Köster, T., Staiger, D. (2014). RNA-Binding Protein Immunoprecipitation from Whole-Cell Extracts. In: Sanchez-Serrano, J., Salinas, J. (eds) Arabidopsis Protocols. Methods in Molecular Biology, vol 1062. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-580-4_35
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DOI: https://doi.org/10.1007/978-1-62703-580-4_35
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