Serial interactome capture of the human cell nucleus.

Thomas Conrad,Sascha Sauer,Anne-Susann Albrecht,Ulf Andersson Ørom,Veronica Rodrigues De Melo Costa,David Meierhofer

doi:10.1038/ncomms11212

Abstract

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present ‘serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)–RNA–protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good 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 signalling 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.

Highlights

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs)
We initially set out to define a nuclear RNA interactome by applying interactome capture technique (IC) to the nuclear and chromatin compartment of K562 myeloid leukaemia cells. We chose this cell line, since growth in suspension culture more allows scaling up starting material for IC by three- to fivefold compared with the two previous whole-cell studies, to compensate for the reduced material obtained from the nuclear compartment[14,15]
Elution of RBP–RNA complexes into low salt buffer enables subsequent enzymatic treatments of IC samples, which can be followed by a second round of oligo d(T)-capture and stringent washes prior to LC-MS/MS detection (Fig. 1a)

Summary

Introduction

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Most nuclear proteins with classic RBDs that were previously identified in HEK293 and HeLa cells by IC are detected by serIC (Fig. 1e)[14,15]. Despite the constraint to the nuclear compartment, the total number of MS/MS peptide identifications is higher in serIC compared with the HeLa RNA interactome data[15], demonstrating high purification efficiency and sensitivity of the experiment (Fig. 2a).

Results

Conclusion