Fig. 5: RNA cross-link rates for RBPs and non-RBPs are both diverse and distinct.

Boxplots show quartiles, center line shows the median, and whiskers show the maxima and minima. a Schematic of the minimal region and total cross-linked RNA. b Definition of terms “cross-link rate” and “complex cross-link efficiency”. c Protein cross-link rates of RBPs (CELF1 n = 6, PCBP1 n = 4, RBFOX1 and STAU1 n = 2, others n= 3). d The percent cross-linking to RNA for the indicated proteins, including only RNA within the minimal region (replicate numbers as above). e Eleven randomly selected proteins that are not known to bind RNA in vivo or in vitro. f Subcellular locations of the randomly selected non-RBPs, as annotated on Uniprot⁵. g UV cross-link rates to RNA for randomly selected, HA-tagged non-RBPs, measuring all RNA (EPB41L5, UBA2, ETS2, and CCIN n = 3, others n = 2). h UV cross-link rates to RNA for randomly selected non-RBPs, including only RNA in the minimal region (EPB41L5, UBA2, ETS2, and CCIN n= 3, others n= 2). i The fraction of easyCLIP reads that map to RNAs of the given category. j Comparison of minimal region cross-link rates between RBPs (maroon) and non-RBPs (EGFP included, violet); vertical line indicates 95% confidence interval. k Minimal region cross-link rates for RBPs with missense mutations in cancer, along with other RBPs, putative RBPs, and non-RBPs. TDRKH is listed as a putative RBP here because the cross-link rate was determined in 293T cells, which do not express the only known target of TDRKH, piRNA. l Minimal region cross-link rate by RBD. Red dots indicate RBPs, purple indicates proteins that may or may not have a direct RNA interaction, and gray dots indicate non-RBPs. m Selected examples of changes in overall RNA binding for recurrent cancer missense mutations in RBPs. n Volcano plot of changes in overall RNA binding for recurrent cancer missense mutations in RBPs. All except PCBP1 are transiently expressed in HEK293T cells. o Cross-linking for selected proteins after stable integration into the genome in the indicated cell type (bars denote mean). p–q Fluorescence polarization assays of bacterially expressed WT and mutant A1CF and KHDRBS2 show ~2-fold increase in affinity for AU-rich RNA. Representative experiments are shown. The experiment was repeated three times with two different protein preparations for each protein pair, all showing a similar increase in affinity for the mutant RBPs. Data are mean ± s.d.