Extended Data Fig. 2: irCLIP-RNP identifies proximal and distal cis-RNA-dependent protein associations (RDAPs).
From: irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein assemblies on RNA
(a) Workflow of irCLIP-RNP to analyse the effects on RNA-dependent protein associations of 1U/µL and 0.02U/µL RNase I treatments in HEK293T. (b) Nitrocellulose images indicate the infrared signal of irCLIP-RNP ligations after 1U/µL and 0.02U/µL RNase I digestions in HEK293T for HNRNPC, HNRNPA2B1, and HNRNPU of n = 2 biologically independent replicates. L: ladder. (c) Rank plots indicate imputed log2-transformed LFQ intensities of proteins detected after HNRNPA2B1/HNRNPC/HNRNPU irCLIP-RNP using two doses of RNase I (1U/µL and 0.02U/µL). Orange dot: UVC-enriched proteins in at least one RNase I dose. (d) Bars indicate the intensity sum of mouse or human unique peptides detected in irCLIP-RNP species-mixing datasets mapping to HNRNPC and HNRNPA2B1 RDAPs determined in c. Orange and purple bars: intensities from unique peptides mapping to either human or mouse RDAP sequences. (e) Volcano plots indicate -log10(P-value) and log2FC values as determined by DEP250 analysis of differential RDAPs between 1U/µL and 0.02U/µL RNase I samples associated with HNRNPC, HNRNPA2B1, and HNRNPU. Black dots: significant proteins; orange circle: selected RDAPs with a significant reduction in 1U/µL RNase I treatment (n = 2 biologically independent experiments). (f) Heatmaps indicate Z scores of imputed log2-transformed LFQ intensities of 1U/µL and 0.02U/µL RNase I samples for the 27 significant RDAPs associating with HNRNPC, HNRNPA2B1, and HNRNPU. Green, orange, and purple boxes: significant RDAPs for HNRNPA2B1, HNRNPC, and HNRNPU, respectively; grey boxes: n.d. values. (g) Bars indicate the overlap between HNRNPC, HNRNPA2B1, and HNRNPU of the 27 significantly reduced RDAPs after 1U/µL RNase I in-lysate digestion. (h) RNA sizing through TBE-Urea gel of HNRNPC irCLIP-RNP isolated RNA in HEK293T from the three RNP subzones ranging from 60–120 kDa (blue), 120–225 kDa (light green), and 225–350 kDa (green). L: ladder. (i) Lines indicate the mean infrared signal distribution of RNA isolated from HNRNPC irCLIP-RNP ligations from three RNP subzones shown in h (n = 2 biologically independent experiments). Data are mean ± SD. Blue line: 60–120 kDa; light-green line: 120–225 kDa; green line: 225–350 kDa RNP subzones.
