High-throughput quantitation of protein–RNA UV-crosslinking efficiencies as a predictive tool for high-confidence identification of RNA-binding proteins

Schematic illustration of method-sensitive free protein recovery in RNA-centric RNA interactome screens. Graphics (A and B) prepared in BioRender. (A) Schematic illustration of non-SILAC LC–MS/MS comparison of RNP fractions isolated independently from UV-crosslinked or non-crosslinked cells by UV-based RNA-centric methods. Volcano plot showing significance values (P-values) of proteins identified in independent RNP fractions by LC–MS/MS as a function of their UV-enrichment ratios. (B) Graphical illustration depicting SRA analysis of RNP fractions (A) and their expected compositions. RNase-insensitive (free) protein exhibiting UV-dependent enrichment would appear as RNA-bound protein during non-SILAC LC–MS/MS comparisons.