Fig. 2

GCLiPP detects cytosolic RBP binding sites with characteristic sequence conservation and structural properties. A Base-pairing probability was calculated for each pair of nucleotides within 200 bp peak called by CLIPper2.0 in Jurkat cells. The average base-pairing matrices for all peaks in the 3′UTR is shown here as a heatmap. B Jurkat RNAseq reads mapped to known RBPs were categorized into different RBDs. Top 10 occurring domains were determined by total reads that can be ascribed to specific domain motif. C Number of RBPs identified through RNA-IC in activated primary human T cells that contain a certain domain. Only top 10 occurring motifs are shown. RBPDB databased was used as a reference for categorizing RBPs in B and C. D Size distribution of n CLIPper-called peaks from datasets of RBP binding detected by GCLiPP, phase-separation methods XRNAX and OOPS, and an amalgamation of 87 RBP eCLIP datasets from ENCORE. µ = mean ± standard deviation. E Sequence conservation of called peaks from D expressed as normalized PhyloP score relative to the peak center. Histograms at bottom show the global average for all peaks for each method. F Normalized PhyloP data from E transformed to display the correlation between eCLIP (y-coordinate) and the indicated methods (x-coordinate) as a function of the distance from peak center. Linear regression statistics and the line of unity (in red) are indicated on each plot. G Genomic snapshots of individual 3′ UTR showing exemplary correlation between TIA1 eCLIP dataset and GCLiPP. GCLiPP is shown in red, while the indicated RBP eCLIP data is shown in blue, and matched control input samples are shown in gray for the 3′ UTRs of the indicated gene. r indicates Pearson correlation between pairs of normalized read density at a given nucleotide for the indicated comparisons. H 2D density plots showing matched correlations between GCLiPP and TIA1 eCLIP (X-axis) and GCLiPP and the matched control input sample (Y-axis) for individual 3′ UTR for all expressed genes in eCLIP and GCLiPP datasets. The t-statistic shown is for a paired t-test of the correlations. I Overlap of CLIPper-called peaks in 3′ UTRs in GCLiPP and eCLIP. Red lines indicate observed overlap of GCLiPP peaks and eCLIP peaks. Gray distribution represents bootstrapped expected overlap, derived by computing overlap of GCLiPP-called peaks with eCLIP peaks shuffled within the same 3′UTR. This analysis was repeated 500 times. The indicated distance represents the number of standard deviations above the mean shuffled overlap of the observed overlap. J Correlation of eCLIP-GCLiPP paired t-tests from H and cytosolic RBP abundance in mRNPs