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

Schematic illustration of S:N-based protein–RNA interaction analysis. Graphics prepared in BioRender. (A) Graphical representation of protein-specific S/N analysis. RNA and protein interact to form an RNP complex; UV-irradiation stabilizes noncovalent RNA–protein interactors to form UV-clRNP. Protein-specific S/N ratios represent the ratio of RNA-bound to unbound counterparts. Background proteins do not contain RNA-bound counterparts. (B) Schematic illustration of SDS-PAGE RNase mobility shift assay (SRA) for determination of protein-specific S/N. The RNase-dependent increase in protein migrating at the expected weight of the unbound counterpart is linearly related to S/N: (|S| + N)_RNase/(N)_untreated = S/N + 1. (C) Schematic illustration of MS-based determination of RNA-bound and unbound (free) protein abundances by TPA. Stacked bar chart showing estimated RNA-bound (%TP_S) and free protein (%TP_N) contributions of three different proteins in the sample. (D) Stacked bar chart showing the difference in RNA-bound and free protein contributions of proteins in RNP fractions isolated by methods with low versus high specificity.