Fig. 2: UBA1^ms leads to a decrease in UBA1 protein in SF3B1^K700E cells.

A Sashimi plots of the UBA1^ms region from total RNA sequencing of SF3B1^WT and SF3B1^K700E K562 cells from previously published data [39]. B qPCR analysis of UBA1^ms relative to 18S in SF3B1^WT and SF3B1^K700E K562 cells (n = 3). Mean ± SEM relative expression. Unpaired t-test. C qPCR analysis of UBA1^WT and UBA1^ms transcript levels in SF3B1^K700E K562 cells after treatment with actinomycin D (ActD) for the indicated time points (n = 3). Results were normalized to 0 h, and MYC was included as a fast-degrading transcript control. Mean ± SEM relative expression, One-phase decay nonlinear curve fit (dotted line). D qPCR analysis of UBA1^WT and UBA1^ms in SF3B1^WT and SF3B1^K700E K562 cells after treatment with cycloheximide (CHX) for 4 h (n = 3). The fraction of UBA1 splice forms is shown within the bars. Mean ± SEM relative expression. Two-way ANOVA with Tukey’s multiple comparisons test. E Agarose gel electrophoresis of the PCR-amplified mis-spliced region of UBA1 (top) and ABCB7 (bottom) in SF3B1^WT and SF3B1^K700E K562 cells. The lower bands correspond to the PCR product of canonically spliced RNA, and the upper bands to mis-spliced RNA. Representative image from three experimental repeats. F Representative polysome profiles of SF3B1^WT and SF3B1^K700E K562 cells recorded at 254 nm. Pooled monosome and polysome fractions are indicated. G qPCR analysis of total UBA1, UBA1^WT, and UBA1^ms RNA transcript distribution in monosome and polysome fractions from SF3B1^WT and SF3B1^K700E K562 cells (n = 5). Housekeeping gene transcript distribution can be found in Supplementary Fig. 2C. Mean ± SEM fraction. Two-way ANOVA with Tukey’s multiple comparisons test. H Immunoblot analysis and I quantification of UBA1 isoforms in whole cell lysates from SF3B1^WT and SF3B1^K700E K562 cells (n = 3). Actin was used as a loading control for total UBA1 and UBA1b; Lamin B1 was used as a loading control for nuclear UBA1a, and relative signals were normalized by lane normalization. Mean ± SEM relative signal intensity. Unpaired t-test with Holm-Šídák’s multiple comparisons test. *, P ≤ 0.05; **, P ≤ 0.01; ns not significant.