Serial interactome capture of the human cell nucleus

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.

Capturing the interactome of newly transcribed RNA

“…In addition, comparison of the 344 RICK-exclusive RBPs with three reported human oligo(dT) capture studies 2,18,19 showed that most of these proteins (295, 85.8%) are truly unique to RICK (Fig. 3b and Supplementary Table 3), so we termed them ‘RICK-unique RBPs’.…”

“…( a ) Hierarchical clustering of the ion intensities of three different LC-MS/MS experiments ( n = 3 biologically independent experiments for both RICK and oligo(dT) capture (with or without EU)), colors in the heatmap indicate the pairwise Pearson correlation between the different data sets ( n = 1,169). ( b ) Venn diagram comparing the 344 RICK-exclusive RBPs and different oligo(dT) capture studies using human cells 2,18,19 . The serial RNA interactome capture (serIC) study by Conrad et al 19 is a human RNA interactome using oligo(dT) capture of the nuclei of K562 cells.…”

“…( b ) Venn diagram comparing the 344 RICK-exclusive RBPs and different oligo(dT) capture studies using human cells 2,18,19 . The serial RNA interactome capture (serIC) study by Conrad et al 19 is a human RNA interactome using oligo(dT) capture of the nuclei of K562 cells.…”

“…( b ) Venn diagram comparing the 295 RICK-unique RBPs and the 914 high-confidence proteins identified by polyA-depleted RICK. The column indicates distribution of the 710 polyA-depleted RICK proteins compared to high-confidence proteins of different oligo(dT) capture studies (green, proteins present in different oligo(dT) capture data sets 1,2,18,19 ; purple, proteins not present in the same oligo(dT) capture data sets). ( c ) Distribution of RNA species identified by METTL1 PAR-CLIP sequencing.…”

SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes

“…The catalog of RBPs is diverse, and has grown to include metabolic enzymes, cell cycle regulators, and many other factors not previously associated with RNA biology (Beckmann et al, 2015; Conrad et al, 2016). As this diverse RBP catalog grows, complex regulatory networks are emerging that rely on highly coordinated and concurrent target association by numerous RBPs.…”