RIP-Chip Analysis: RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling
Post-transcriptional regulation of gene expression plays an important role in complex cellular processes. Just like transcription factors regulate gene expression through combinatorial binding to multiple, physically dispersed cis elements, mRNA binding proteins can regulate the translation of functionally related gene products by coordinately binding to subsets of mRNAs. The networks of mRNA binding proteins that facilitate this fine-tuning of gene expression are poorly understood. By combining genomic technologies with standard molecular biology tools, we have helped pioneer the development of high-throughput technologies for the global analysis of subsets of mRNAs bound to RNA-binding proteins. This technique is termed RIP-Chip and stands for RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling. This approach is also referred to as “ribonomic profiling” and has revealed valuable information about the workings of mRNP networks in the cell and the regulation of gene expression. In this chapter, we describe the latest advances that we have made in the RIP-CHIP technology.
Key wordsPost-transcriptional gene regulation Ribonomics RIP-Chip RNA-binding Protein (RBP) Immunoprecipitation (IP) microarray microarray expression profiling array systems biology
We would like to acknowledge the expert technical help from David Frank and Marcy Kuentzel of the microarray core, Center for Functional Genomics, University at Albany-SUNY and input from the other Tenenbaum Lab members. This work was supported in part by NIH grant U01HG004571 to SAT from the NHGRI.
- 10.Kato, T., Hayama, S., Yamabuki, T., Ishikawa, N., Miyamoto, M., Ito, T., Tsuchiya, E., Kondo, S., Nakamura, Y., Daigo, Y. (2007) Increased expression of insulin-like growth factor-II messenger RNA binding protein-1 is associated with tumor progression in patients with lung cancer. Clin Cancer Res 13, 434–442.CrossRefPubMedGoogle Scholar
- 16.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., Warren, S. T. (2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107, 477–487.CrossRefPubMedGoogle Scholar
- 32.Duttagupta, R., Tian, B., Wilusz, C. J., Khounh, T. J., Soteropoulos, P., Ouyang, M., Dougherty, J. P., Peltz, S. W. (2005) Global analysis of Pub1p targets reveals a coordinate control of gene expression through modulation of binding and stability. Mol Cell Biol 25, 5499–5513.CrossRefPubMedGoogle Scholar
- 35.Zhang, L., Ding, L., Cheung, T. H., Dong, M. Q., Chen, J., Sewell, A. K., Liu, X., Yates, J. R., 3rd, and Han, M. (2007) Systematic identification of C. elegans miRISC proteins, miRNAs and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. Mol Cell 28, 598–613.CrossRefPubMedGoogle Scholar
- 55.Yang, J., Chennathukuzhi, V., Miki, K., O’Brien, D. A., Hecht, N. B. (2003) Mouse testis brain RNA-binding protein/translin selectively binds to the messenger RNA of the fibrous sheath protein glycerldehyde-3-phosphate-dehydrogenase-S and suppresses its translation in vitro. Biol Reprod 68, 853–859.CrossRefPubMedGoogle Scholar