Figure 6.

Distinct RNA dynamics in erythroid differentiation intensify SF3B1^mt mis-splicing. A, Proportional Venn diagram of genes undergoing statistically significant AS [FDR < 0.001, absolute difference in percentage spliced in levels (Abs. ΔPSI) > 0.2] in the HSPC (green; MDS-RS CD34⁺ vs. NBM CD34⁺), nucleated erythroid [red; MDS-RS RS vs. NBM EB, Erythroid (N)] and anucleate erythroid [blue; MDS-RS Siderocytes vs. NBM Ret^PB, Erythroid (A)] populations. B, Gene Ontology biological process (GO BP) enrichment analysis results comprising genes mis-spliced in both the HSPC and erythroid (N) populations are shown in the top bar chart. The Human Gene Atlas enrichment results with the lowest adjusted P value are shown in the bottom table. C, Frequency of AS events split by rMATS category in each sample group comparison. SE, skipped exon; RI, retained intron; MXE, mutual exon exclusion; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice site. Statistical comparisons of A3SS+RI frequencies were performed with Fisher exact test. D, Box plots of percent spliced-in (PSI) values of literature-validated SF3B1^mt-induced A3SS events in MDS-RS samples, separated by sample type (CD34, GPA, RS). Known targeting by NMD is indicated with a red circle; in-frame events without a PTC are indicated by a blue circle. E, Box plots of PSI values in newly identified ASEs affecting known driver genes implicated in the pathogenesis of MDS and CSA. Known targeting by NMD is indicated with a red circle; PTC detection with unverified NMD is indicated with an orange circle; in-frame events without a PTC are indicated by a blue circle. F, Distribution of base pair distances from cryptic A3SS sites to canonical splice sites (horizontal axis) in HSPC and Erythroid (N). Further detail is provided in −400 bp to 0 bp for increased contrast. Lines at −30 bp and −10 bp demarcate the interval associated with SF3B1 mis-splicing (38). Additional lines at −140 bp and −330 bp demarcate additional intervals of interest. G, Sequence logos of canonical and A3SS sequences encompassing the 3′ splice site (starting at −35 bp upstream of the AG motif) and statistical comparison through a two-sample logo. H, Frequency of A3SS events per rMATS cell type comparison where the splice site shift remains in-frame (blue) or induces a frameshift event (orange). I, Frequency of exon insertion events per rMATS cell-type comparison where the splice site shift incorporates a new PTC (pink) or remains in-frame with no PTC induction (green). J and K, RNA velocity analysis of transcriptomically identified HSPC and erythroblast subsets in 10x scRNA-seq, visualizing the percentage of spliced transcripts along pseudotime in the total cell populations (violin plots) or separated by sample group (scatter plots). The analysis in J includes all transcripts, whereas K excludes ribosomal and globin transcripts. L, UMAP overlay of FACS-purified HSPC subsets and GPA+ EB from 1 SF3B1^mt MDS-RS patient after Smart-seq3xpress (SS3x), visualizing true versus predicted cell-type identity. M, RNA velocity analysis of spliced RNA read percentages in the FACS-sorted SS3× experiment, analyzed independently of the 10x dataset. This graph excludes ribosomal and globin transcripts. N, Mean (±SEM) differences in PSI after 3 hours cycloheximide treatment (70 μg/mL) versus DMSO (1:1,000, vehicle) in MDS-RS CD34 and GPA cells. SF3B1^mt-associated NMD-targeted ASEs with sufficient coverage are shown at far left, SF3B1^mt-associated in-frame ASEs at middle-left, and endogenous NMD-targeted transcripts at middle-right. The far-right plot visualizes all ASEs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonstatistically significant.