Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses (original) (raw)
- STAVROULA MILI and
- JOAN A. STEITZ
- Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06536-9812, USA
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.
Footnotes
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.7151404.
- Accepted August 10, 2004.
- Received July 29, 2004.
Copyright 2004 by RNA Society