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. Author manuscript; available in PMC: 2021 Jun 23.
Published in final edited form as: Nat Struct Mol Biol. 2020 Aug 17;27(10):989–1000. doi: 10.1038/s41594-020-0477-6

Fig. 3 ∣. Integration of eCLIP and RNA-seq data defines regulatory classes of RBPs and transcripts.

Fig. 3 ∣

a,b, Volcano plots showing the distribution of fold changes in transcript levels upon modulation of destabilizers (a) and stabilizers (b), with distribution histograms shown at the top. a, Depletion of DDX6 (left) and overexpression of SNRPA (right). b, Depletion of UBAP2L (left) and overexpression of BOLL (right). Transcripts with log2 (fold change) ≥ 1.23 and FDR-corrected P ≤ 0.05 from two replicates are in color, with red and green denoting, respectively, transcripts with or without at least one significant RBP binding peak (≥4-fold enrichment and P ≤ 10−2 versus SMInput in two eCLIP replicates, Fisher’s exact test for read numbers <5 or χ2test for read numbers ≥5). c,d, Bar plots showing the percentage overlap between genes significantly up- or downregulated (log2 (fold change) ≥ 1.23 and FDR-corrected P ≤ 0.05 in two replicates) and significantly bound (≥4-fold enrichment and P ≤ 10−2 versus SMInput in two eCLIP replicates) upon knockdown (KD) or overexpression (OE) of candidate destabilizers (c) and stabilizers (d) (*P < 0.01, ***P < 10−3, ****P < 10−4, hypergeometric test versus nontargeting shRNA or FLAG overexpression, as appropriate). Data for graphs in c and d are available in Extended Data Fig. 3d-f. e–h, Cumulative distribution plots of transcript log2-transformed fold changes of overexpression versus vector control or shRNA-mediated knockdown versus nontargeting control, as indicated, for the destabilizers DDX6 (e) and SNRPA (f) and the stabilizers UBAP2L (g) and BOLL (h). Distributions are shown for transcripts with the indicated significant read enrichments over SMInput (P ≤ 10−2) from eCLIP analysis (4- to 8-fold, green; 8- to 16-fold, red) or transcripts that are not significantly bound (‘Not bound’, P > 10−2 or <4-fold enrichment, gray). n is the number of genes; P values are given versus ‘Not bound’, two-tailed Mann–Whitney U test from two replicates. i,j, Genome browser views from shRNA-mediated knockdowns showing RNA-seq reads (shRNA knockdown, maroon; nontargeting shRNA control, pink) and eCLIP reads (IP, blue; SMInput, gray) for PARN at the RPS21 locus (i) and CLK3 at the NELFCD locus (j). The y axes denote read density in RPM.