Extended Data Fig. 10: UPF1 supports HNRNPC RNA splicing surveillance of EGF-regulated proliferation mRNAs.
From: irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein assemblies on RNA
(a) Workflow of HNRNPC and UPF1 irCLIPv2 and HNRNPC-UPF1 Re-CLIP in EGF-stimulated (0, 15, 30, and 60 min) A431 cells. (b-c) Nitrocellulose images indicate the infrared signal of HNRNPC and UPF1 irCLIPv2 ligations in EGF-stimulated (0, 15, 30, and 60 min) A431 cells in b and infrared signal of HNRNPC-UPF1 Re-CLIP ligations from EGF-stimulated (0, 30, and 60 min) A431 cells in c. The signal represents pooled libraries of n = 2 biologically independent experiments. (d) Pie chart indicates the percentage of 1721 common AS genes categorized as protein-coding (orange), lncRNA (yellow), or pseudogene (green). (e) Stacked bar graph indicates the percentage of the 2388 common AS regions residing in protein-coding genes categorized as 5’UTR (orange), 3’UTR (yellow), or CDS (green). (f) Heatmap indicates the percentage of RT stop location of HNRNPC and UPF1 irCLIPv2 and HNRNPC Re-CLIP followed by UPF1 in the genomic features 5’ and 3’ UTRs, exon, and intron of the AS transcripts. (g) Workflow of HNRNPC and UPF1 irCLIPv2 after UPF1 and HNRNPC KD in EGF-stimulated (0, 60 min) A431 cells. (h) Nitrocellulose images indicate the infrared signal of HNRNPC and UPF1 irCLIPv2 ligations after UPF1-KD or HNRNPC-KD, respectively (n = 2 biologically independent experiments). (i) Bars indicate KD efficiencies of HNRNPC and UPF1 in EGF-stimulated HNRNPC- and UPF1-KD irCLIPv2 samples, as detected by qPCR. Data are represented as mean (n = 2 biologically independent experiments). (j) Scatter plots indicate the log2 mean HNRNPC and UPF1 total signal across 3’UTR regions and the Z score estimated from log2FC upon UPF1 or HNRNPC-KD during EGF stimulation. Marginal box plots indicate the distribution of co-bound HNRNPC and UPF1 regions in AS transcripts or not (red and orange dots) or not co-bound regions in AS transcripts or not (blue and grey dots). Boxes indicate the interquartile range (IQR) with 25th and 75th percentile box limits; whiskers denote the 1.5 × IQR from the 25th and 75th percentiles; center lines: medians; black dots: outliers; red dots: mean values. P-value = 3.6e-3**, P-value = 1.0e-4***; P-value = 0.32 not significant, P-value = 0.02*, P-value = 2.1e-3, P-value = 0.95 not significant – one-way ANOVA with two-sided Dunnett’s multiple comparison test against not co-bound regions (grey dots) (n = number of 3’UTR regions specified in the plot). (k) Scatter plots indicate the Z score values estimated against control siRNA in HNRNPC and UPF1 irCLIPv2 knockdown data of the 30 EGF-responsive 3’UTRs of RI transcripts co-BR by HNRNPC and UPF1 at 0 and 60 min EGF stimulations. Blue lines and grey bands: fitted linear regression with standard error. (l) De-novo motifs discovered in EGF-responsive co-bound and co-regulated (co-BR) 3’UTRs of RI transcripts. (m) Volcano plot indicates the -log10(FDR) and max(log2FC) values as determined by DESeq262 analysis of DE genes in bulk RNAseq from EGF-treated (0, 15, 30, and 60 min) A431 cells. Orange and blue dots: up and downregulated genes. (n) Heatmap indicates the Z score of normalized counts of 10 co-BR RI genes by HNRNPC and UPF1 showing time-dependent-response to EGF stimulation. Orange and blue boxes: up and downregulated genes (o) IGV tracks indicate normalized coverage of HNRNPC and UPF1 irCLIPv2 timecourse/knockdown and HNRNPC-UPF1 Re-CLIP samples at 3’UTR of DDX3X. Tracks show the signal sum of two replicates. Shades of blue boxes: EGF stimulation time points. (p) De novo structure prediction using AlphaFold 3 and manual refinement provides a three-dimensional model of the protein-RNA multimer complex comprising HNRNPC, UPF1, and 3’ UTR mRNA of DDX3X. (q) Representative immunoblot image of n = 3 independent experiments indicates HNRNPC and UPF1 protein signal after native RNA pull-down of biotinylated RND3 and DDX3X 3’UTR binding regions in 1 h EGF-stimulated A431 cells. (r) Bars indicate fold change against scramble control of HNRNPC and UPF1 protein signal after native RNA pull-down in 1 h EGF-stimulated A431 cells. Data are mean ± SD; UPF1-DDX3X P-value = 0.015*, UPF1-RND3 P-value = 7.6e-3**, HNRNPC-DDX3X P-value = 0.014*, HNRNPC-RND3 P-value = 0.016* – unpaired two-tailed Student’s t-test against scramble control 1 and 2 (n = 3 biologically independent experiments).
