Blood Relatives: Splicing Mechanisms underlying Erythropoiesis in Health and Disease

Abstract

During erythropoiesis, hematopoietic stem and progenitor cells transition to erythroblasts en route to terminal differentiation into enucleated red blood cells. Transcriptome-wide changes underlie distinct morphological and functional characteristics at each cell division during this process. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA-the most highly expressed transcript in erythroblasts-was the focus of many seminal studies on the mechanisms of pre-mRNA splicing. We now understand that pre-mRNA splicing plays an important role in shaping the transcriptome of developing erythroblasts. Recent advances have provided insight into the role of alternative splicing and intron retention as important regulatory mechanisms of erythropoiesis. However, dysregulation of splicing during erythropoiesis is also a cause of several hematological diseases, including β-thalassemia and myelodysplastic syndromes. With a growing understanding of the role that splicing plays in these diseases, we are well poised to develop gene-editing treatments. In this review, we focus on changes in the developing erythroblast transcriptome caused by alternative splicing, the molecular basis of splicing-related blood diseases, and therapeutic advances in disease treatment using CRISPR/Cas9 gene editing.

Keywords: CRISPR/Cas; RNA-seq; erythropoiesis; globin; intron retention; myelodysplastic syndrome (MDS); nonsense-mediated decay (NMD); pre-mRNA splicing; spliceosome; ß-thalassemia.

Figure 1.. Changes in gene expression and splicing occur during terminal erythroid differentiation.

Erythropoiesis is characterized by changes in cell morphology, including nuclear size, color (due to hemoglobinization), and chromatin condensation, which are coordinated with changes in gene expression. During terminal erythroid differentiation, cells progress from proerythroblasts (PRO), to basophilic erythroblasts (EARLY and LATE BASO), to polychromatophilic erythroblasts (POLY), to orthrochromatic erythroblasts (ORTHO), before enucleation to become red blood cells (RBCs) (also called reticulocytes). In human erythroblasts, a subset of genes is downregulated—some top associated Gene Ontology terms are shown to the right (purple line)—while a subset of genes is concurrently upregulated (green line). Changes in splicing occur in the later stages of erythropoiesis (mostly from late baso to ortho), including increased alternative splice site usage and intron retention.

Figure 2.. Single-nucleotide mutations in key regulatory regions of the β-globin gene disrupt expression in β-thalassemia.

Point mutations in varying regions of the β-globin gene are shown schematically, and the frequencies of these mutations listed in the HbVar database ( http://globin.bx.psu.edu/hbvar/menu.html) are shown in brackets at the left. RNA polymerase is shown in yellow. Gene regions are divided into a) promoter, b) splice sites, c) other intronic regions, d) exons, and e) polyadenylation site. These mutations (red X’s) have varying effects, illustrated below each example, but all lead to decreased or abolished expression of the β-globin transcript. AS, alternative splicing; NMD, nonsense-mediated decay; PTC, premature termination codon.

Figure 3.. Driver mutations in myelodysplastic syndromes introduce single amino acid changes to core splicing factors.

Somatic mutations in general splicing factors U2AF1, SRSF2, and SF3B1 have been implicated in myelodysplastic syndromes. Although these proteins are involved in the recognition of splice site sequences in all pre-mRNAs, tissue-specific splicing defects are observed in blood cells. These mutations lead to aberrant splicing in erythroblasts in a wide range of transcripts. ESE, exonic splicing enhancer; SS, splice site.