Splicing Control in Normal and Aberrant Erythropoiesis

Alternative splicing is employed by all eukaryotic cells to increase proteome diversity and to regulate gene expression. RNA sequencing analysis of purified populations of erythroblasts at different stages of maturation has led to the identification of a dynamic alternative splicing program that directly modulates the protein isoform expression of cytoskeletal proteins and genes involved in RNA processing, heme biosynthesis, and iron transport. Regulated interactions of multiple RNA-binding proteins and cis-regulatory sequences located within exons or their flanking introns promote or inhibit functional spliceosome assembly at splice junctions, leading to altered exon inclusion or intron retention. Exon skipping regulates tissue and stage specific isoform expression of red cell membrane cytoskeletal proteins including EPB41, ankyrin, and band 3. Intron retention can lead to a frame shift during translation and introduction of a premature termination codon (PTC), that marks the transcript for degradation via the nonsense mediated decay pathway (NMD) upon export from the nucleus into the cytoplasm. Intron retention leading to posttranscriptional regulation of gene expression during terminal erythroid maturation has been identified in genes involved in RNA processing and iron transport including SF3B1, SNRNP70, SLC25A37 and SLC25A28. Mutations that alter mRNA splice sites or introduce PTCs lead to a variety of congenital anemias including beta thalassemia, hereditary pyropoikilocytosis, hereditary elliptocytosis, and hereditary spherocytosis. Aberrant mRNA splicing has subsequently been shown to lead to acquired anemias in subsets of patients with myelodysplastic syndromes (MDS). Somatic missense mutations in components of the spliceosome are the most common category of mutations in MDS. These point mutations lead to changes in the RNA binding specificity of the involved proteins and aberrant splicing of a subset of transcripts. Mutant SF3B1, the most commonly mutated splicing factor in MDS, has been shown to cause aberrant pre-mRNA splicing and an increase in transcripts predicted to undergo NMD due to use of upstream, cryptic 3' splice sites. Our group and others evaluating the strong genotype-phenotype association between SF3B1 point mutations and subtypes of MDS with ring sideroblasts have shown that the expression of the mitochondrial iron transporter, ABCB7, is decreased in samples from SF3B1-mutant MDS patients due to cryptic 3' splice site selection and introduction of a PTC between exons 8 and 9. The identification and functional validation of additional aberrantly spliced mutant-SF3B1 target genes is ongoing, with the goal of understanding how point mutations in a core component of the mRNA splicing machinery can lead to such specific effects on erythroid maturation.