Serial interactome capture of the human cell nucleus
Novel RNA-guided cellular functions are paralleled by an increasing number of RNA binding proteins (RBPs). We present “serial interactome capture” (serIC), a multiple purification procedure of UV-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with maximal specificity. We apply serIC to nuclei of proliferating K562 cells to obtain the first human nuclear interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including fewer factors without known RNA binding domains that are in better agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signaling and double strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs. The nuclear interactome presented here is a stepping-stone towards deciphering of the functional RNA-protein network in the mammalian nucleus.
Sample Processing Protocol
UV crosslinking, cells lysis, nuclei isolation, high LiDS lysis, poly(A)RNA-RBP-capture with oligo(dT)beads, DNAse digest, recapture, LC-MS/MS
Data Processing Protocol
Samples were analyzed by MaxQuant according to the details in the paper.
Conrad T, Albrecht AS, de Melo Costa VR, Sauer S, Meierhofer D, Ørom UA. Serial interactome capture of the human cell nucleus. Nat Commun. 2016 Apr 4;7:11212 PubMed: 27040163