Journal: bioRxiv

Article Title: All exons are not created equal - Exon vulnerability determines the effect of exonic mutations on splicing

doi: 10.1101/2023.06.14.544306

Figure Lengend Snippet: Overlapping motifs indicate exon vulnerability. A . K-mer analysis (jellyfish) was carried out on the sequences of the exons that were either vulnerable or resilient, and differentially enriched hexamers were compared between the two groups. Shown are the top enriched hexamers in the vulnerable (blue) and resilient (green) exons. Significantly enriched k-mers were identified using a proportion test and adjusting for multiple testing with Bonferroni. B . 11 mutations affecting the GAAGAA motif, and 17 mutations creating a TAGG ESS motif was introduced in 15-mer biotinylated RNA oligonucleotides, and the wild type (WT) and mutant (MUT) sequences was analyzed with surface plasmon resonance imaging (SPRi) using recombinant SRSF1 and hnRNPA1. Binding is shown as Log2(Rmax). Red lines indicate the two mutations in ATM and MFSD8 . Wilcoxon paired test was performed between WT and MUT binding scores. * p ≤ 0.05, ns p > 0.05. C . ENCODE eCLIP data from K562 cells against SRSF1 and hnRNPA1 was plotted across the last 50 bp of the upstream exon, the first and last 300 bp of the upstream intron, the first and last 50 bp of the resilient vulnerable and control exons, the first and last 300 bp of the downstream intron, and the first 50 bp of the downstream exon. Control is the mean binding of all exons in the three groups.

Article Snippet: Affinity purification of RNA-binding proteins was performed as previously described ( ) using biotinylated RNA oligonucleotides (LGC biosearch technologies, Risskov, Denmark, sequences in supplements) immobilized on Dynabeads M-280 streptavidin magnetic beads (Invitrogen) and incubated with HeLa cell nuclear extract.

Techniques: Mutagenesis, SPR Assay, Imaging, Recombinant, Binding Assay