Fig. 1: RNA sequencing reveals UBA1^ms in an iPSC model of MDS-SF3B1.

A Origin of iPSC lines and experimental overview. B Representative flow cytometry diagrams of SF3B1^WT and SF3B1^K700E iPSC-derived cells after 12 days of hematopoietic differentiation (left) and another 14 days of erythroid culture (right). Colored gates indicate cells used in downstream analyses. C Sashimi plots of the mis-spliced region of UBA1 in SF3B1^WT and SF3B1^K700E from total RNA sequencing of iPSC-derived GlyA⁺ erythroblasts (n = 1). Black, canonical splice junction counts; orange, mis-spliced junction counts. y-axis, absolute read counts. D qPCR analysis of UBA1^ms relative to 18S in CD34⁺ HSPCs (n = 4) and E erythroblasts (n^WT = 6; n^K700E = 7) derived from SF3B1^WT and SF3B1^K700E iPSCs. Mean ± SEM relative expression. Unpaired t-test. F Agarose gel electrophoresis of the PCR-amplified exon 5-6 mis-spliced region of UBA1 in iPSC-derived CD34⁺ cells (n = 4). The lower band corresponds to the PCR product of canonically spliced, and the upper band to mis-spliced UBA1. G Immunoblot analysis and H quantification of UBA1a/b protein levels in iPSC-derived CD34⁺ cells (n = 4). Actin was used as a loading control and relative signals were normalized by lane normalization factor. Mean ± SEM relative UBA1 signal intensity. Unpaired t-test with Holm-Šídák’s multiple comparisons test. *, P ≤ 0.05; ***, P ≤ 0.001. The numbers (#) below the blot indicate experimental repeats. VAF variant allele frequency, PSI percent spliced-in, RNA seq RNA sequencing, bp base pairs.