Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes

Abstract

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

Figure 1

Properties of A3SS misregulated in SF3B1-mutant compared with wild-type SF3B1 MDS HSCs. (a) Sequence logos for upstream and downstream cryptic 3′ splice sites along with their associated canonical sites. (b) Density plot showing distance (log2) between pairs of 3′ splice sites. Blue line: upstream cryptic. Red line: downstream cryptic. Green line: A3SS unaffected by SF3B1 mutation. (c) 3′ splice site strengths (maximum entropy) for upstream and downstream control (unregulated) A3SS, upstream cryptic sites and their associated canonical sites, and downstream cryptic sites and their associated canonical sites. (d) BP scores. (e) Distance of highest scoring predicted BP from associated 3′ splice site. (*P<0.05, **P<0.01).

Figure 2

Validation and semi-quantification of cryptic 3′ splice site usage in five selected genes from the rMATS analysis in SF3B1-mutant MDS HSC samples. (a) PCR products of the five genes (TMEM14C, ENOSF1, DYNLL1, SEPT6 and HINT2) were amplified from SF3B1-mutant MDS samples (Mut), wild-type MDS samples (WT) and healthy control samples (HC) run on an Agilent 2100 Bioanalyzer instrument using the DNA 1000 kit. The aberrant transcripts (indicated by the higher band) were observed in the SF3B1-mutant MDS samples. (b) Semi-quantification of the PCR bands showed high cryptic to canonical isoform ratio in the SF3B1-mutant MDS samples.

Figure 3

Cryptic 3′ splice site usage of ABCB7 in SF3B1-mutant MDS HSCs. (a) Visualization of RNA-Seq traces for the ABCB7 gene (intron 8-exon 9 junction) in two healthy controls, two MDS patients with no known splicing factor mutations and two SF3B1-mutant MDS cases, using Integrative Genomics Viewer (IGV). A cryptic 3′ splice site is observed in the ABCB7 gene between exon 8 and exon 9 in the SF3B1-mutant MDS cases, leading to an addition of 21 base pairs to the coding sequence causing a premature termination codon at the seventh amino acid (indicated with *). (b) Sanger sequencing traces of gel extracted bands corresponding to the canonical ABCB7 transcript and to the aberrant ABCB7 transcript containing the additional 21 base pairs (highlighted in blue).

Figure 4

Aberrant splicing of ABCB7 in HSCs and cultured erythroblast from SF3B1-mutant MDS samples, in the K562-SF3B1^K700E cell line and a SF3B1-mutant pancreatic cell line. (a) RT-PCR confirmation of aberrant splicing of ABCB7 in SF3B1-mutant MDS HSC samples. The higher 174 bp band corresponds to the aberrant ABCB7 transcript and the lower 153 bp band corresponds to the canonical ABCB7 transcript. The aberrant ABCB7 transcript (higher 174 bp band) was observed in the SF3B1-mutant MDS samples (Mut), but not in samples from wild-type MDS patients (WT) or in samples from healthy controls (HC). (b) Aberrant splicing of ABCB7 in cultured erythroblast from SF3B1-mutant MDS patients. Each panel shows data from one different SF3B1-mutant MDS patient (Mut) and from one different healthy control (HC). RT-PCR for ABCB7 was performed on cultured erythroblasts at day 11 and day 14 of culture treated with and without the NMD inhibitor cycloheximide (CHX). The higher 174 bp band corresponds to the aberrant ABCB7 transcript and the lower 153 bp band corresponds to the canonical ABCB7 transcript. The RT-PCR results showed an increase in the product corresponding to the aberrant ABCB7 transcript (higher 174 bp band) in the SF3B1-mutant patient samples treated with CHX compared with untreated samples. No aberrant ABCB7 splicing was seen in the samples from healthy controls. (c) Aberrant splicing of ABCB7 in K562-SF3B1^K700E. RT-PCR for ABCB7 was performed on cultured K562-SF3B1^K700E and K562-SF3B1^WT cells treated with and without the NMD inhibitor CHX. The higher 174 bp band corresponds to the aberrant ABCB7 transcript and the lower 153 bp band corresponds to the canonical ABCB7 transcript. The RT-PCR results showed an increase in the product corresponding to the aberrant ABCB7 transcript (higher 174 bp band) in the K562-SF3B1^K700E cells treated with cycloheximide (CHX) compared with the untreated cells. No aberrant ABCB7 splicing was seen in the K562-SF3B1^WT cells untreated or treated with CHX. (d) Aberrant splicing of ABCB7 in Panc 05.04 cells with SF3B1 mutation (K700E). RT-PCR for ABCB7 was performed on cultured Panc 05.04 cells treated with and without the NMD inhibitor CHX. The higher 174 bp band corresponds to the aberrant ABCB7 transcript and the lower 153 bp band corresponds to the canonical ABCB7 transcript. The RT-PCR results showed an increase in the product corresponding to the aberrant ABCB7 transcript (higher 174 bp band) in the Panc 05.04 cells treated with CHX compared with the untreated cells.