Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells

H Dolatshad, A Pellagatti, M Fernandez-Mercado, B H Yip, L Malcovati, M Attwood, B Przychodzen, N Sahgal, A A Kanapin, H Lockstone, L Scifo, P Vandenberghe, E Papaemmanuil, C W J Smith, P J Campbell, S Ogawa, J P Maciejewski, M Cazzola, K I Savage, J Boultwood, H Dolatshad, A Pellagatti, M Fernandez-Mercado, B H Yip, L Malcovati, M Attwood, B Przychodzen, N Sahgal, A A Kanapin, H Lockstone, L Scifo, P Vandenberghe, E Papaemmanuil, C W J Smith, P J Campbell, S Ogawa, J P Maciejewski, M Cazzola, K I Savage, J Boultwood

Abstract

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34(+) cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicing/processing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34(+) cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Figures

Figure 1
Figure 1
Effects of SF3B1 knockdown in myeloid cell lines. Each cell line transfected with siRNA targeting SF3B1 was compared with the corresponding cell line transfected with the scramble control. (a) SF3B1 mRNA expression measured 3 days after knockdown. (b) Growth curves of cells with SF3B1 knockdown, compared with cells transfected with the scramble control, as assessed by trypan blue exclusion. (c) Cell cycle analysis of cell lines following SF3B1 knockdown. (d) Erythroid differentiation in myeloid cell lines with SF3B1 knockdown treated with 50 μm hemin, as measured by HBG1 and KLF1 expressions relative to the scramble control. (e) Percentage of CD36+CD71+ and CD71+CD235a+ populations in K562 cells with SF3B1 knockdown compared with the scramble control. Results in subpanels a–d were obtained from scramble n=2 and SF3B1 siRNA as follows: a, n=3; b, n=3; c, n=3 for SKM1 and TF1, n=6 for HEL and n=9 for K562; d, n=3 for TF1 and HEL, n=6 for K562. Results in subpanel e were obtained from scramble n=4 and SF3B1 siRNA n=4. *P<0.05.
Figure 2
Figure 2
Effects of SF3B1 knockdown on gene expression and splicing. (a) ABCB7 and FTMT expression levels in TF1, K562, HEL and SKM1 cells with SF3B1 knockdown, as measured by qRT-PCR 48 h post transfection. Each cell line transfected with siRNA targeting SF3B1 was compared with the corresponding cell line transfected with the scramble control. (b) Reverse transcription-PCR of TP53 exons 5–7 showing aberrant splicing in K562 cells with SF3B1 knockdown (siRNA) compared with scramble (SCR). (c, d). qRT-PCR analysis using primers that monitor general gene expression (GE) in a constitutive exon (Ex4 of CCNA2 and Ex3 of STK6) or primers specific for splice junctions corresponding to exon inclusion or skipping in the cyclin A2 (CCNA2) and Aurora Kinase A (STK6) genes in K562 cells. Cells with SF3B1 knockdown (siRNA) show alternative splicing events. Results in subpanel a were obtained from scramble n=3 and SF3B1 siRNA n=4. Results in subpanel c–d were obtained from scramble n=2 and SF3B1 siRNA n=3. *P<0.05.
Figure 3
Figure 3
Examples of genes showing significant differential exon usage between MDS patients with SF3B1 mutation in comparison with control, obtained from RNA-Seq data analysis using DEXSeq. The graphs show some of the top ranking genes with significant differential exon usage. The exons highlighted in purple represent the significant differential exon usage.
Figure 4
Figure 4
Examples of genes showing significant differential exon usage in MDS patients with SF3B1 mutation in comparison with wild type, obtained from RNA-Seq data analysis using DEXSeq. The graphs show some of the top ranking genes with significant differential exon usage. The exons highlighted in purple represent the significant differential exon usage.

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