Figure 1.

A novel splicing isoform of SF3B1, SF3B1ins, is detected in sideroblastic myelodysplastic syndromes (MDS-RS) patients and myeloid cell lines expressing mutant SF3B1. (A) Representation of the aberrantly spliced junction of SF3B1. (B) 3D modeling of the eight amino acid insertion in the H3 repeat of SF3B1 Huntingtin, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1 (HEAT) domain. The site of insertion and the lysine in position 700 are indicated in both normal (top) and predicted (bottom) structures. Primary sequences of normal and aberrant H3 repeat (amino acids 588–605) are indicated below the 3D structures. The inserted sequence is colored in orange. (C) Detection of SF3B1ins transcript in mononuclear cells from myelodysplastic syndromes (MDS) patients harboring SF3B1 mutations. RNA was extracted from mononuclear cells of subjects with normal bone marrow (lanes 1–4), from MDS patients without RS (lanes 5–10) and from MDS-RS patients. The mutational status of SF3B1 is indicated for MDS-RS patients, as follows: mut—mutated; WT—wild-type. RT-PCR was performed using primers allowing specific detection of SF3B1ins or detection of both aberrant (upper band) and canonical (lower band) transcripts of SF3B1, ENOSF1 and TMEM14C. (°: ENOSF1 RT-PCR on patient 2 could not be performed due to an insufficient quantity of material); (D,E) Inducible expression of SF3B1^K700E in K562 cells and UT-7 cells generates SF3B1ins transcript. RT-PCR was performed from K562 cells (D) and UT-7 (E), expressing SF3B1^WT (left) or SF3B1^K700E (right) (I: induced; NI: non induced), using primers as described in C. Steady-state SF3B1 protein level was achieved by Western blot (Figure S1); (F) Detection of SF3B1ins transcript in K562 transiently expressing distinct SF3B1 variants. RT-PCR was performed using primers as described in C. Data information: In (D,E), representative results of at least three independent RT-PCR experiments are presented. L: ladder.