Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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 419:68–91
Chiaruttini C, Milet M, Hayes DH, Expert-Bezancon A (1989) Crosslinking of ribosomal proteins S4, S5, S7, S8, S11, S12 and S18 to domains 1 and 2 of 16S rRNA in the Escherichia coli 30S particle. Biochimie 71:839–852
Darnell RB (2010) HITS-CLIP: panoramic views of protein–RNA regulation in living cells. Wiley Interdiscip Rev RNA 1:266–286
Galgano A, Gerber AP (2011) RNA-binding protein immunopurification-microarray (RIP-chip) analysis to profile localized RNAs. Methods Mol Biol 714:369–385
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 141:129–411
Harvey I, Garneau P, Pelletier J (2002) Forced engagement of a RNA/protein complex by a chemical inducer of dimerization to modulate gene expression. Proc Natl Acad Sci U S A 99:1882–1887
Jain R, Devine T, George AD, Chittur SV, Baroni TE, Penalva LO, Tenenbaum SA (2011) RIP-chip analysis: RNA-binding protein immunoprecipitation-microarray (chip) profiling. Methods Mol Biol 703:247–263
Jensen KB, Darnell RB (2008) CLIP: crosslinking and immunoprecipitation of in vivo RNA targets of RNA-binding proteins. Methods Mol Biol 488:85–98
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.
Lee JE, Lee JY, Wilusz J, Tian B, Wilusz CJ (2010) Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells. PLoS One 5:e11201
Niranjanakumari S, Lasda E, Brazas R, Garcia-Blanco MA (2002) Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26:182–190
Ryder SP, Recht MI, Williamson JR (2008) Quantitative analysis of protein-RNA interactions by gel mobility shift. Methods Mol Biol 488:99–115
Sokoloski KJ, Wilusz J, Wilusz CJ (2008) The preparation and applications of cytoplasmic extracts from mammalian cells for studying aspects of mRNA decay. Methods Enzymol 448:139–163
Sokoloski KJ, Dickson AM, Chaskey EL, Garneau NL, Wilusz CJ, Wilusz J (2010) Sindbis virus usurps the cellular HuR protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells. Cell Host Microbe 8:196–207
Tenenbaum SA, Lager PJ, Carson CC, Keene JD (2002) Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. Methods 26:191–198
Urlaub H, Kühn-Hölsken E, Lührmann R (2008) Analyzing RNA-protein crosslinking sites in unlabeled ribonucleoprotein complexes by mass spectrometry. Methods Mol Biol 488:221–245
Acknowledgements
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this chapter
Cite this chapter
Palusa, S., Wilusz, J. (2012). Approaches for the Identification and Characterization of RNA-Protein Interactions. In: Dinman, J. (eds) Biophysical approaches to translational control of gene expression. Biophysics for the Life Sciences, vol 1. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-3991-2_10
Download citation
DOI: https://doi.org/10.1007/978-1-4614-3991-2_10
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4614-3990-5
Online ISBN: 978-1-4614-3991-2
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)