Fig 1: AQR KD recapitulates ∼40% of SF3B1MUT-specific cryptic 3′ss events and induces SUGP1 mRNA missplicing(A) Hierarchical clustering and heatmap analysis of the usage of 123 core events in AQR KD samples and corresponding control samples. Rows represent the 123 core events, while columns represent the samples.(B) Scatterplot displaying the ΔPSI values for the 123 core events in AQR KD samples compared with SF3B1MUT samples. The horizontal axis shows the ΔPSI value between SF3B1MUT and control samples, while the vertical axis shows the ΔPSI value between the AQR KD sample and control sample. The pie plot (upper left) indicates the proportion of 3′ss events recapitulated (brown) or not recapitulated (blue) by AQR KD.(C) IGV plots of cryptic 3′ss event recapitulated by AQR loss in samples with SF3B1 mutations, SUGP1 loss, AQR loss, or WT. PSI values are labeled for each track.(D) Western blotting of protein extracts for SUGP1 from either CRISPR-engineered K562 cells with WT (W) or K700E (K) SF3B1 or parental K562 cells treated with a negative control siRNA (siC) or one of two independent siRNAs targeting AQR.(E) RT-PCR of cryptic 3′ss (white arrow) and canonical 3′ss (black arrow) produced from splicing of the indicated transcripts.(F) IGV plots of cryptic 3′ss usage in SUGP1 intron 6 in WT, SF3B1K700E, SUGP1 KO, and AQR KD samples. PSI values are shown for each track.(G) Scatterplot representation of differentially spliced 3′ss changes on U2 complex genes between AQR KD and controls showing the magnitude (difference of PSI; x axis) and significance (−log10(q value); y axis).(H) RT-PCR of the cryptic 3′ss and canonical 3′ss produced from splicing of SUGP1 intron 6. Black arrowheads indicate the canonical 3′ splice site (canonical 3′ss), and the white arrowheads indicate the cryptic 3′ splice site (crytpic 3′ss).(I) Same RT-PCR as in (H) but with or without emetine treatment. Black arrowheads indicate the canonical 3′ splice site (canonical 3′ss), and the white arrowheads indicate the cryptic 3′ splice site (crytpic 3′ss).(J) Scatterplot depicting the correlation between AQR KD and SUGP1 KO on the SF3B1MUT-specific events recapitulated by AQR KD. The horizontal axis shows the PSI changes between SUGP1 KO and control samples, while the vertical axis shows the PSI changes between AQR KD and control samples. The indicated R value and p value were determined by Pearson correlation.