The Erythroid Intron Retention Program Encompasses Developmentally Stable and Dynamic Networks and Regulates Diverse Gene Classes

Computational analysis of RNA-seq data from highly purified human erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in proerythroblasts, but elevated intron retention in orthochromatic erythroblasts prior to enucleation. Dynamic regulation of late erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues.