Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses.
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.
Mili
Stavroula
S
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, 295 Congress Ave., Box 9812, New Haven, Connecticut 06536-9812, USA.
Steitz
Joan A
JA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D011233:Precipitin Tests; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D016601:RNA-Binding Proteins