Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses
Open Access
- 23 September 2004
- journal article
- Published by Cold Spring Harbor Laboratory in RNA
- Vol. 10 (11), 1692-1694
- https://doi.org/10.1261/rna.7151404
Abstract
Immuno- and other affinity-purification approaches are commonly used to characterize the composition of ribonucleoprotein complexes. While associations detected by these procedures are often interpreted as reflecting in vivo interactions, it is also possible that they arise from reassociation of molecules after cell lysis. Here we used an experimental approach that allowed us to distinguish between these possibilities. Surprisingly, we show that the association of the RNA-binding protein HuR with its target mRNA, c-fos, as detected by co-immunoprecipitation, results largely from reassociation of molecules subsequent to cell lysis. The existence of such postlysis reassortments thus demonstrates that co-immunoprecipitation does not always recapitulate the in vivo state of ribonucleoprotein complexes.Keywords
This publication has 8 references indexed in Scilit:
- Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arraysMethods, 2002
- HuR and mRNA stabilityCellular and Molecular Life Sciences, 2001
- HuR binding to cytoplasmic mRNA is perturbed by heat shockProceedings of the National Academy of Sciences of the United States of America, 2000
- Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the invivo stability of ARE-containing mRNAsThe EMBO Journal, 1998
- RNA stabilization by the AU-rich element binding protein, HuR, an ELAV proteinThe EMBO Journal, 1998
- Quantitative Measurement of mRNA Using the RNase Protection AssayMethods in molecular biology (Clifton, N.J.), 1994
- Immunological methods for purification and characterization of heterogeneous nuclear ribonucleoprotein particlesPublished by Elsevier BV ,1990
- [32] Immunoprecipitation of ribonucleoproteins using autoantibodiesMethods in Enzymology, 1989