HASPIN depletion dysregulates RNA splicing in AML. (A) Bar plot revealing replicate multivariate analysis of transcript sequences quantification of significant splicing events induced on shRNA-mediated HASPIN depletion in Kasumi-1 cells. Major splicing patterns (left) are as follows: A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; MXE, mutually exclusive exon; RI, retained intron; SE, skipped exon. Events that had at least 50 junction-spanning reads, FDR ≤0.05, and absolute delta percent spliced in (|dPSI|) ≥0.10 were considered significant. (B) Heat map depicting GO enrichments of significantly spliced events for splicing pattern. Terms and event type were grouped by hierarchical clustering as indicated by column and row dendrograms. Cell color represents –log10(P value) for corresponding GO term enrichment. (C) Venn diagram of genes with differential alternative splicing events (DAS genes) and genes with differential expression (DE genes) on HASPIN depletion in Kasumi-1 cells. Intersection represents genes that are both DAS and DE on HASPIN depletion. Significant splicing events or differentially expressed genes were overlapped based on respective cutoffs. (D) Proportional bar plots reporting changes between transcript functional biotypes in significant splicing events of each pattern on HASPIN depletion in Kasumi-1 cells. Notation is “Initial:Final.” Each color corresponds with a transition between biotypes. Transcript biotypes assigned according to ENSEMBL transcriptome annotation. “Unannotated” indicates that the transcript was not located in the annotation database. (E) Sashimi plot (left) and reverse transcription polymerase chain reaction (RT-PCR) validation (right) of BCL2L11 SE events observed on HASPIN depletion in Kasumi-1 cells. Sashimi plot depicts change in mean percent spliced in (PSI) levels of target exons between control (blue) and HASPIN knockdown (red) in Kasumi-1 cells. Inclusion and exclusion form depicted in black schematic beneath plots. Data are grouped from 3 biological replicates. RT-PCR analysis of BCL2L11 target exon inclusion in Kasumi-1 cells expressing control (C) or HASPIN-targeting shRNA 1 (1). Schematic of inclusion (larger) and skipping (smaller) isoforms included to left of gel image. Percent exon inclusion between the 2 isoforms after HASPIN depletion is quantified. Data are mean ± SD of 4 independent experiments. Statistical significance determined by unpaired 2-tailed Student t test. ∗∗P < .01. (F) RBP motif enrichment analysis of SRSF1 and HNRNPC in significant SE splicing events on HASPIN depletion in Kasumi-1 cells. Motif enrichment (top) and P value (bottom) are reported at various positions along representative cassette exon for splicing events with significantly promoted exon inclusion (orange, 2641 events) or suppressed exon inclusion (purple, 2498 events) on HASPIN depletion relative to nondifferentially spliced background (green, 7049 events) as determined by rMAPS2. A region of 250 bp was analyzed using a 50 bp sliding window.
