Fig. 5.

A global snapshot of RNPs in HEK293 cells. a Schematic of the experimental setup: HEK293 cells were UV-cross-linked using 0 (noCL), 0.015 (dark red), 0.15 (red) and 1.5 (dark yellow) J/cm² 254 nm light in triplicates. Total RNA from input (whole-cell lysate) and PTex-purified samples were analysed by RNA-Seq. b Deletions in RNA from input and PTex samples; frequency of mutations in transcripts correlate with higher UV doses. Boxplot centre line represents median, bounds are first and third quantile, and whiskers extend to 1.5 times the inter-quantile range (n = 3 biologically independent experiments except for input 0.015 J/cm² (n = 2). c Mutations (deletions = green, substitutions = blue) enriched in UV-irradiated samples were plotted to their position relative to AUG and Stop codon in coding sequences and serve as indicator for protein-binding sites. Note that we cannot delineate which protein bound to which position. Plots for PTex are shown; for input see Supplementary Fig. 17, 18. d–g Input (whole-cell lysate) and PTex-purified sample were analysed by label-free mass spectrometry. d Volcano plots of proteins enriched by PTex (FDR 0.01) under the three cross-linking conditions. e Overlap of PTex-enriched proteins (enriched in all 9 replicates, FDR 0.01) is 3037 (these PTex proteins are from here on coloured in orange). f Protein abundance (IBAQ intensities of input samples) does not correlate with PTex enrichment (log2-fold change of intensities [CL/-CL]). g PTex does not select for a subset of proteins based on general features such as molecular weight, pI or hydrophobicity. Boxplot centre line represents median, bounds are first and third quantile, and whiskers extend to 1.5 times the inter-quantile range. h, i PTex of individual predicted RNA-associated proteins. ATP-binding cassette sub-family F member 2 (ABCF2) and T-complex protein 1 subunit eta (CCT7) have not been reported to bind RNA. Both are enriched after PTex in a UV-irradiation-dependent fashion, indicating that they indeed associate with RNA in vivo. For full blots see Supplementary Figure 19