Fig. 5: Discovery of in vivo STRAP-binding RNA targets by eCLIP-seq.

a Schematic illustration of the overall experimental design, showing that STRAP binds with ³²P-labeled RNAs (Bottom); the eCLIP method was used to establish sequencing libraries (Right). The schematic diagram was created by the authors. b Pie charts displaying peak distributions of enriched read density within STRAP eCLIP. The fraction of STRAP peaks, defined from eCLIP-seq (P ≤ 10⁻³ and ≥ 5-fold, relative to INPUT), locates along the different genic regions across the mouse transcriptome. Each biological replicate shows a highly comparable percentage for indicated categories between replicates. c, d Metagene plots showing STRAP binding frequency on pre-mRNA in two pooled embryos (E9.0; n = 7 per pool) (c) or two biologically replicated WT EBs (9-day-old) (d). The X-axis indicates a composite intro-exon-intron boundary, containing sequences for 300 nt in the upstream intron and the first and last 100 nt of the exon and 300 nt in the downstream intron. STRAP eCLIP crosslink site density around constitutive 5′ and 3′ splice sites normalized by respective input density is plotted on the Y-axis. The dash line boxes show amplified regions for peak enrichment. See “Methods” section for further details. e Left, logo visualization of the top HOMER motif outputs generated from the merged eCLIP dataset. Right, the fraction of target regions and respective P-value with each motif are displayed. f Binding of STRAP with Nnat motif-containing RNA oligonucleotides. Purified GST-STRAP or GST was incubated with biotin-labeled WT or MUT or 200-fold excess non-labeled RNA oligos as indicated. The complexes were separated on 6% polyacrylamide native gel. The experiment was repeated three times independently with similar results. g BubbleMap visualization of representative GO functions for STRAP target genes through RNA-protein interaction.