Approaches for the Identification and Characterization of RNA-Protein Interactions
Based on analyses of conventional RNA binding motifs, there are well over 550 RNA binding proteins produced in living cells. Thus a detailed understanding of the mechanisms of post-transcriptional regulation requires an understanding of how these trans-acting factors act in both local and combinatorial fashions. The goal of this chapter is to provide a roadmap for approaching the identification of RNA–protein interactions and how to decipher fundamental aspects of their roles in the regulation of gene expression. Many of the techniques outlined below are applicable to the study of RNA–RNA interactions as well. Collectively they should provide a rational approach to gain mechanistic insights into a variety of post-transcriptional processes.
KeywordsSodium Dodecyl Sulfate Cold Phosphate Buffer Saline Sepharose Bead Streptavidin Agarose TEDS Buffer
We wish to thank Ashley Neff, Stephanie Moon, Jerome Lee, John Anderson, and other members of the Wilusz laboratories for helpful comments and critical suggestions. Related RNA–protein work in the laboratory is supported by NIH grant GM072481 to J.W.
- Beach DL, Keene JD (2010) Ribotrap:targeted purification of RNA-specific RNPs from cell lysates through immunoaffinity precipitation to identify regulatory proteins and RNAs. Methods Mol Biol 419:68–91Google Scholar
- Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M Jr, Jungkamp AC, Munschauer M, Ulrich A, Wardle GS, Dewell S, Zavolan M, Tuschl T (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–411PubMedCrossRefGoogle Scholar
- Kishore S, Jaskiewicz L, Burger L, Hausser J, Khorshid M, Zavolan M (2011) A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nat Methods. doi: 10.1038/nmeth.1608.