RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation
RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation
复制标题
DOI：
10.1261/rna.565207
复制
发表时间：
2007-06-01
期刊：
影响因子：
4.5
通讯作者：
Collins, Kathleen
中科院分区：
文献类型：
--
作者：
Hogg, J. Robert;Collins, Kathleen
最近的研究发现了非编码 RNA (ncRNA) 的意想不到的多样性，尽管这些研究对其生物学意义的了解有限。已经开发了许多用于鉴定和表征蛋白质相互作用的通用方法，但缺乏用于表征细胞 ncRNA 相互作用的类似方法。在这里，我们描述了 RNA 亲和串联 (RAT)，这是一种原始的、完全基于 RNA 标签的方法，用于亲和纯化内源组装的 RNP 复合物。我们通过分离体内组装在 RNA 聚合酶 II 或 III 转录的 ncRNA 上的 RNP 来证明 RAT 的通用性。使用 RAT 结合质谱法和蛋白质 - RNA 相互作用测定的蛋白质鉴定，我们定义并表征了内源组装的人 7SK RNP 的先前未预料到的蛋白质亚基。我们发现 7SK RNA 存在于具有不同蛋白质组成和对细胞应激反应不同的 RNP 混合群体中。新鉴定的 7SK RNP 成分 hnRNP K 的耗尽会改变 7SK RNA 在不同 RNP 之间的分配。我们的结果确立了基于 RNA 的通用 RNP 亲和纯化方法的实用性，并提供了对 7SK RNP 动力学的深入了解。
Recent studies have uncovered an unanticipated diversity of noncoding RNAs (ncRNAs), although these studies provide limited insight into their biological significance. Numerous general methods for identification and characterization of protein interactions have been developed, but similar approaches for characterizing cellular ncRNA interactions are lacking. Here we describe RNA Affinity in Tandem (RAT), an original, entirely RNA tag-based method for affinity purification of endogenously assembled RNP complexes. We demonstrate the general utility of RAT by isolating RNPs assembled in vivo on ncRNAs transcribed by RNA polymerase II or III. Using RAT in conjunction with protein identification by mass spectrometry and protein - RNA interaction assays, we define and characterize previously unanticipated protein subunits of endogenously assembled human 7SK RNPs. We show that 7SK RNA resides in a mixed population of RNPs with different protein compositions and responses to cellular stress. Depletion of a newly identified 7SK RNP component, hnRNP K, alters the partitioning of 7SK RNA among distinct RNPs. Our results establish the utility of a generalizable RNA-based RNP affinity purification method and provide insight into 7SK RNP dynamics.