Extended Data Fig. 3: Confirmation of RBP knockdown/overexpression, numbers of genes differentially regulated/unaffected by RBP perturbation, and region-level analysis of bound transcripts.
From: Large-scale tethered function assays identify factors that regulate mRNA stability and translation
a,b, shRNA-mediated depletion of RBPs in HEK293T cells using 3-5 distinct shRNAs for each RBP, as indicated, compared to nontargeting shRNA control. a, Western blots with GAPDH or tubulin serving as loading controls, as indicated. b, Bar graphs indicating RBP transcript levels determined by RT–qPCR, normalized to levels of 18S rRNA. Data are shown as mean ± SD (n = 3 replicates). Asterisks denote significance at P < 0.05 determined by two-sided Student’s t-test. c, Overexpression of RBPs in HEK293T cells. Bar plots showing transcript levels (RPKM) for each RBP following transfection of RBP expression constructs or FLAG vector control. Data are shown as mean ± s.d. (n = 2 replicates). d–g, Numbers of up- or downregulated (log2-fold change ≥ 1.23 and FDR-corrected P ≤ 0.05) or unchanged genes for transcripts bound (≥4-fold enriched and P ≤ 10−2 versus eCLIP SMInput) or not bound by the indicated RBP for knockdown (d) and overexpression (e) of destabilizing RBPs and for knockdown (f) and overexpression (g) of stabilizing RBPs. h,i, Volcano plots showing the distribution of fold changes in transcript levels, with distribution histograms at the top, upon depletion of the destabilizer PARN (h) and depletion of the stabilizer CLK3 (left) and overexpression of the destabilizer IFIT2 (right) (i). Transcripts with log2(fold change) ≥1.23 and FDR-corrected P ≤ 0.05 are in color, with red and green denoting transcripts with or without at least one significant RBP binding peak (≥4-fold enriched and P ≤ 10−2 versus SMInput in eCLIP), respectively. j, Heatmap showing significance in differential expression of genes significantly differentially expressed (log2(fold change) ≥ 1.23 and FDR-corrected P ≤ 0.05) and significantly bound (≥4-fold enriched and P ≤ 10−2 versus SMInput in eCLIP) versus all unbound genes upon knockdown (KD) or overexpression (OE) of candidate RBPs in each region. Significance was calculated using a two-tailed Mann-Whitney U test. Uncropped images for a are available as source data online, and data for graphs in b, c are available as source data online.
