Figure 2: Identification of U-rich sequences and stem–loop secondary structure as recognition elements of RC3H1.

(a) Log10 frequencies of 7mers occurring in the 41-nucleotide (nt) window around the RC3H1 preferred crosslink sites are shown for 4SU-2 PAR-CLIP and 6SG PAR-CLIP libraries. U-rich sequences are frequently occurring in both 4SU and 6SG libraries. (b) Enrichment of indicated 7mers in the 41-nt window around the RC3H1 (left) or IGF2BP1 (right) preferred crosslink sites compared with all 3′UTR sequences. U-rich sequences with A contents are specifically enriched in RC3H1 3′UTR-binding sites, whereas 7mers containing known IGF2BP1 motif (CAU) are enriched in IGF2BP1 3′UTR-binding sites to the similar degree. (c) A heat map showing the coverage of 7mers, indicated on the left, around the preferred crosslinks in 3′UTR RC3H1 consensus binding sites. U-rich elements with A contents and CDE are indicated. U-rich sequences are found in the close vicinity of crosslink sites, which is indicative of direct association of RC3H1 with U-rich sequences. (d) Enrichment of indicated stem–loop structures in the 41-nt window around the RC3H1 (left) or IGF2BP1 (right) preferred crosslink sites compared with 41 nt sequences randomly selected from the 3′UTRs of target transcripts as a background model. Various stem–loop structures (n-m-n indicates a hairpin structure of n-mer stem and m-mer loop) are enriched in RC3H1 3′UTR-binding sites but not in 3′UTR IGF2BP1-binding sites. (e) The seed alignment and consensus structure of motifs 1 and 2 are shown. ARE, AU-rich elements.