Fig. 2: INSCRIBE identifies authentic RBFOX2 binding sites through confident C-to-U edit clusters. | Nature Communications

Fig. 2: INSCRIBE identifies authentic RBFOX2 binding sites through confident C-to-U edit clusters.

From: High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues

Fig. 2

a Sanger sequencing result of the in vitro RNA editing assay validated the C-to-U editing enzymatic activity of the recombinant APOBEC1-nanobody (APO1-Nb). UTR: untranslated region. b Integrative genome viewer (IGV) tracks showing RBFOX2 eCLIP peaks (HEK293T) on the target gene APP expanding a 1500 bp window, compared with the read coverage, SAILOR-quantified edit fraction and FLARE-determined edit clusters of three replicates of RBFOX2-INSCRIBE, along with the enzyme-only negative controls. The confident edit clusters derived from three replicates were also shown. c HOMER de novo motif discovery identified the canonical RBFOX2 motif UGCAUG as the top motif in all RBFOX2-INSCRIBE replicates, using a cumulative hypergeometric distribution for p values. d Distribution of the distance between the closest UGCAUG and confident edit clusters for RBFOX2-INSCRIBE replicates (orange) and confident edit clusters (violet), enzyme-only control replicates (light blue) and confident edit clusters (dark blue), and RBFOX2-STAMP (green). A peak at 0nt distance indicated the enrichment of UGCAUG in proximity to the edit cluster center, demonstrating the RBFOX2-binding driven editing. The RBFOX2-INSCRIBE confident edit clusters presented much stronger enrichment of adjacent UGCAUG motif than the individual replicates. e, f The actual fraction of UGCAUG-containing RBFOX2-INSCRIBE edit clusters/RBFOX2 eCLIP IDR peaks (dots) compared to the 20 permuted clusters (box plot, n = 20). Z-scores: RBFOX2-INSCRIBE, 49.93; Enzyme-only control, 0.43; RBFOX2-STAMP, 23.08; RBFOX2-eCLIP, 117.83. f The actual fraction of eCLIP-overlapping RBFOX2-INSCRIBE edit clusters (diamonds) compared to the permuted clusters (box plot, n = 20). Z-scores: RBFOX2-INSCRIBE, 18.84; Enzyme-only control, 0.39; RBFOX2-STAMP, 5.45. In (e–f), the box shows the quartiles while the whiskers extend to show the rest of the distribution; the median is represented by the center line. Enrichment is defined as the ratio of actual fraction to the mean of the permuted-derived fractionThe actual fraction was plotted using the confident edit clusters derived from 3 INSCRIBE technical replicates. g Distribution of the distance between the closest eCLIP peak and confident edit clusters for RBFOX2-INSCRIBE (violet) and enzyme-only control (light blue).

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