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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Cancer Discov. 2021 Oct 6;12(2):522–541. doi: 10.1158/2159-8290.CD-20-1513

Figure 4. ZRSR2 mutations promote specific mis-splicing events.

Figure 4.

a. Intron retention events in BPDCNs with a ZRSR2 mutation (n=2, red) compared pairwise to normal pDCs (n=2, noted as samples A and B) and to those events in BPDCNs with no known splicing factor mutation (n=4, gray). b. Density plots of frequencies of U2-type (blue) and U12-type (red) introns among aberrantly retained introns (Fisher’s exact test p-value ≤ 0.05) in pairwise analyses of ZRSR2 mutant versus wild-type BPDCNs and patient-derived xenografts (PDXs). N=2 ZRSR2 mutant and n=4 splicing factor wild-type in each. Dotted lines represent ΔMSI −0.2 and +0.2. c. RNA-seq reads of an aberrantly retained U12 intron (intron 4) in human DERL3 from BPDCN PDXs with or without ZRSR2 mutation (equivalent scale for all samples). d. Western blot for ZRSR2 and beta-actin in CAL1 cells (parental or ZRSR2 knockout) and in BPDCN PDXs with wild-type or mutant ZRSR2. e. Western blot from CAL1 cells at baseline (day 0) and at 2 and 7 days after doxycycline induction of non-targeting control or ZRSR2-targeted shRNAs. f. Mis-splicing events after knockdown of ZRSR2 from cells shown in panel (e), calculated by pairwise comparison between ZRSR2 knockdown and controls (n=3 biological replicates each) on days 0, 2, and 7 of doxycycline induction. The number (and percentage) of events on each day is depicted in a single bar colored by different types of mis-splicing events: intron retention (red), 3’ cryptic splice site (green), and exon skip (blue). g. Density plots of frequencies of U2-type (blue) and U12-type (red) introns in pairwise analysis of CAL1 with ZRSR2 knockdown versus control on day 7 after the doxycycline induction (Fisher’s exact test p-value ≤ 0.05), plotted as in (b). h. (left) RNA-seq reads and RT-PCR from CAL1 cells after the induction of control or ZRSR2-targeted shRNA showing retention of DERL3 intron 4. (right) Number of DERL3 intron 4 RNA-seq reads plotted for control and ZRSR2 knockdown cells at days 0, 2, and 7 after doxycycline shRNA induction (n=3 biological replicates each, groups compared by t test).