Fig. 3

Elavl1a prefer to bind single-stranded RNA in vivo and in vitro which enriched in structurally variable regions in 3′ UTRs. a Scatter plot shows the significance and occurrence of RNA-binding motif enriched in structurally variable windows at 3′ UTR between 4 h.p.f. and 6 h.p.f.; P values were calculated by Fisher’s exact test. Inner pie chart shows 47.1% of transcripts with structurally variable regions at their 3′ UTR containing Elavl1 binding motif. b Scatter plot shows Elavl1a’s enrichment in UV (+) sample at 4 h.p.f.. LFQ, label free quantitation. c Distribution of Elavl1a peaks across the length of mRNA and binding motif identified by Dreme (MEME suite) with Elavl1a-binding peaks in 3′ UTR (E-value = 1.8 × 10^− 332). d icSHAPE metaprofile around Elavl1a binding sites and unbound sites with the same motif shows that Elavl1a tend to bind ssRNA in vivo.e The structure models of six endogenous RNA probes containing Elavl1a binding sites. Elavl1a binding sites were colored in red background. f Demonstration of endogenous Elavl1a pulled down by endogenous RNA probes containing Elavl1a binding sites. Upper, western blotting; lower, quantification level. Error bars, mean ± s.d., n = 3. P values were calculated using Student’s t test. g Demonstration of purified Flag-Elavl1a pulled down by endogenous RNA probes containing Elavl1a binding sites. Upper, western blotting; lower, quantification level. Error bars, mean ± s.d., n = 3. P values were calculated using Student’s t test. h The structure models of designed P1 wild-type, P1 mutant, and P1 rescue RNA probes containing Elavl1a binding sites and flanking regions. i Demonstration of endogenous Elavl1a pulled down by designed endogenous RNA probes containing Elavl1a binding sites. Upper, western blotting; lower, quantification level. Error bars, mean ± s.d., n = 3. P values were calculated using Student’s t test. j Demonstration of purified Flag-Elavl1a pulled down by designed endogenous RNA probes containing Elavl1a binding sites. Upper, western blotting; lower, quantification level. Error bars, mean ± s.d., n = 3. P values were calculated using Student’s t test. k EMSA (left) and line graph quantification (right) showing the binding ability of purified Flag-Elavl1a with designed P1 wild-type, P1 mutant, and P1 rescue RNA probes containing Elavl1a binding sites. In total, 100 nM of RNA probes was incubated with different concentrations of Flag-Elavl1a protein. The RNA binding ratio was calculated by (RNA protein) / ((free RNA) + (RNA protein)). Error bars, mean ± s.d., n = 3