title: RNA-binding protein immunopurification-microarray (RIP-Chip) analysis to profile localized RNAs.
abstract: Post-transcriptional gene regulation is largely mediated by RNA-binding proteins (RBPs) that modulate mRNA expression at multiple levels, from RNA processing to translation, localization, and degradation. Thereby, the genome-wide identification of mRNAs regulated by RBPs is crucial to uncover post--transcriptional gene regulatory networks. In this chapter, we provide a detailed protocol for one of the techniques that has been developed to systematically examine RNA targets for RBPs. This technique involves the purification of endogenously formed RBP-mRNA complexes with specific antibodies from cellular extracts, followed by the identification of associated RNAs using DNA microarrays. Such RNA-binding protein immunopurification-microarray profiling, also called RIP-Chip, has also been applied to identify mRNAs that are transported to distinct subcellular compartments by RNP-motor complexes. The application and further development of this method could provide global insights into the subcellular architecture of the RBP-RNA network, and how it is restructured upon changing environmental conditions, during development, and possibly in disease.
authors: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
authors: Alessia
authors: A
authors: Galgano
authors: André P
authors: AP
authors: Gerber
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity; D002457:Cell Extracts; D003116:Color; D004492:Edetic Acid / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008863:Microspheres; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D034443:RNA Transport; D016372:RNA, Antisense / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000378:metabolism; D013997:Time Factors
chemical_list: D000906:Antibodies; D002457:Cell Extracts; D005456:Fluorescent Dyes; C029152:G-substrate; D009419:Nerve Tissue Proteins; C471242:PUM1 protein, human; C471243:PUM2 protein, human; D016372:RNA, Antisense; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D013205:Staphylococcal Protein A; D012313:RNA; D012685:Sepharose; D004492:Edetic Acid