Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses
Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses
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DOI：
10.1261/rna.7151404
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发表时间：
2004-11-01
期刊：
影响因子：
4.5
通讯作者：
Steitz, JA
中科院分区：
文献类型：
--
作者：
Mili, S;Steitz, JA
免疫和其他亲和纯化方法通常用于表征核糖核蛋白复合物的组成。虽然通过这些方法检测到的关联通常被解释为反映了体内的相互作用，但它们也可能是由细胞裂解后分子的重新结合引起的。在这里，我们使用了一种实验方法，使我们能够区分这些可能性。令人惊讶的是，我们发现通过共免疫沉淀检测到的rna结合蛋白HuR与其靶mRNA c-fos的结合主要是由于细胞裂解后分子的重新结合。因此，这种裂解后重组的存在表明，共免疫沉淀并不总是概括核糖核蛋白复合物的体内状态。
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 post-lysis reassortments thus demonstrates that co-immunoprecipitation does not always recapitulate the in vivo state of ribonucleoprotein complexes.