RNA-interactome capture in human embryonic stem cells (hESCs)

Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carried out a proteome-wide screen and identified 810 proteins that directly bind RNA in hESCs. We determined the RBP catalog by using RNA-interactome capture (RIC), a method based on UV light-mediated cross-linking (CL) of RNAs to proteins in living cells, followed by oligo(dT) purification of poly(A)-RNA-protein complexes and mass spectrometry analysis of captured proteins. As control, we applied a similar strategy to non-cross-linked (non-CL) samples. To uncover the identity of the eluted proteins, we performed in-solution tryptic digestion of CL and non-CL eluates and analyzed their contents by a high-resolution mass spectrometer (Q-Exactive Plus). We then performed differential proteome analysis between CL and non-CL eluates, resulting in a set of 810 high-confidence protein groups, defined as the hESC RNA-interactome. RIC was carried out in four independent biological replicates. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells".