A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis

Abstract

Alternative pre-messenger RNA splicing remodels the human transcriptome in a spatiotemporal manner during normal development and differentiation. Here we explored the landscape of transcript diversity in the erythroid lineage by RNA-seq analysis of five highly purified populations of morphologically distinct human erythroblasts, representing the last four cell divisions before enucleation. In this unique differentiation system, we found evidence of an extensive and dynamic alternative splicing program encompassing genes with many diverse functions. Alternative splicing was particularly enriched in genes controlling cell cycle, organelle organization, chromatin function and RNA processing. Many alternative exons exhibited differentiation-associated switches in splicing efficiency, mostly in late-stage polychromatophilic and orthochromatophilic erythroblasts, in concert with extensive cellular remodeling that precedes enucleation. A subset of alternative splicing switches introduces premature translation termination codons into selected transcripts in a differentiation stage-specific manner, supporting the hypothesis that alternative splicing-coupled nonsense-mediated decay contributes to regulation of erythroid-expressed genes as a novel part of the overall differentiation program. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells.

Figure 1.

Morphology of differentiation stage-specific erythroblast populations purified by FACS as described previously (18).

Figure 2.

Flow chart showing the process by which major alternative splicing events were detected in discrete populations of erythroblasts at specific stages of their terminal differentiation.

Figure 3.

RT-PCR validation of alternative splicing events in human erythroblasts. Shown are amplification products containing (filled circles) or lacking (empty circles) alternative exons amplified from erythroblast transcripts. Gene names are indicated above each lane.

Figure 4.

Differentiation stage-specific alternative splicing switches in human erythroblasts. (A) Alternative exons that exhibit stage-specific upregulation in splicing efficiency. Gels show splicing changes assessed by RT-PCR using primers located in flanking constitutive exons. Amplification products from proE RNA and orthoE RNA are shown in the left and right lanes, respectively. Gene name is indicated above each gel, while the calculated PSI is shown below each lane. Arrowheads indicate exon inclusion products. Asterisk indicates genes for which PCR results indicate larger splicing changes than were predicted bioinformatically. (B) Alternative splicing event that exhibits a differentiation-associated decrease in PSI as cell matures. (C) A differentiation-associated switch involving mutually exclusive exons of 100 and 91 nt, respectively. The size difference allows electrophoretic separation of the alternative products.

Figure 5.

Stage-specificity of splicing switches during erythroblast differentiation. (A) Splicing efficiency of selected splicing switches across erythroblast populations. (B) Broader analysis of alternative exons exhibiting a splicing switch during erythroblast maturation. Number of exons with |ΔPSI| > 25 is represented by bar height at the indicated stage transitions.

Figure 6.

Differential expression of full-length and PTC transcript isoforms in differentiating erythroblasts. (A) RNA binding protein genes that increase expression of PTC isoforms in orthoE by exon inclusion (above) or by exon skipping (below). Exons in red contain stop codons (indicated by asterisks). PSI values for the PTC exons in proE and orthoE are shown at the right. (B) Genes in which PTC isoforms are more abundant in proE (above) or are not regulated in late erythroblasts (below).

Figure 7.

Validation of stage-specific PTC splicing events in human erythroblasts. (A) Transcripts in which PTC isoforms are more abundant in late erythroblasts due to upregulation of PTC-exons; (B) transcripts in which PTC transcripts are relatively more abundant in early erythroblasts due to exon skipping events that alter translational reading frame. Gene names and differentiation stage are indicated above each lane, while calculated PSI values are shown below each lane. Arrowheads indicate PCR bands representing PTC isoforms. * indicates genes for which PCR results indicate larger splicing changes than were predicted bioinformatically.

Figure 8.

Effects of NMD inhibition on expression of PTC splicing events. (A) PTC transcripts produced by exon inclusion events were assayed in proE-enriched day 9 and polyE/orthoE-enriched day 16 erythroblast cultures without (−) or with (+) cycloheximide treatment. Cycloheximide dramatically increased the expression of PTC isoforms, especially at day 16, as expected if their abundance in untreated cells was limited by active NMD machinery. For SNRNP70, PCR detects the smaller of the two PTC exons depicted in Figure 7A, B. Cycloheximide effects on PTC-upon-exon skipping events. As in (A), cycloheximide increased expression of PTC isoforms, as expected, if their abundance in untreated cells was limited by NMD. The higher proportion of full-length transcripts predicted in late erythroblasts was maintained ±NMD inhibition. Gene names and culture stage are indicated above each lane, while PSI values shown below each lane were measured by densitometric analysis of stained gels. Arrowheads indicate PCR bands representing PTC isoforms.