This SHINE II proposal will address a novel mechanism of gene regulation, intron retention (IR), as it applies to a major splicing regulator / blood disease gene (SF3B1), and an iron homeostasis gene, SLC25A28 (mitoferrin-2). These gene models represent developmentally dynamic and developmentally stable retention events, respectively, that likely are regulated by different mechanisms. Goals of this proposal are to define the mechanism(s), and to obtain proof of principle that this mechanistic information can lead to methods for modulation of retention in a potentially therapeutic manner. IR transcripts, which by definition retain at least one unspliced intron, represent an abundant fraction of many genes' transcriptional output in human erythroblasts: up to 50% in both SF3B1 and SLC25A37 (mitoferrin-1). The latter is a close paralog of SLC25A28 and is very highly expressed in late erythroblasts, but SLC25A28 is more amenable to study due to smaller intron size. IR can theoretically impose major post-transcriptional limits on expression of translatable mRNA during normal development, and by extension mis-regulated could effect quantitative abnormalities of expression in putative `intron retention' diseases. Indeed, aberrant intron retention is a hallmark of myelo- dysplasia syndrome (MDS) in patients with ZRSR2 mutations. Little is known about molecular mechanisms controlling IR. IR in SF3B1 is developmentally dynamic, being up-regulated from 20% to 50% as cells mature from proerythroblasts (lower IR) to orthochromatophilic erythroblasts (higher IR). One model for IR regulation in this gene involves activity of `decoy' or `cryptic' exons in the intron that may interact with the flanking splice sites to block intron excision. In contrast, IR is SLC25A28 (and SLC25A37) is already high in proerythroblasts and remains high throughout erythroblast differentiation. Preliminary data show that an antisense morpholino directed against a distal intron region can alter IR in the endogenous SLC25A28 gene, indicating the presence of an intron splicing enhancer that potentially could act via formation of an RNA bridge.
The aim of this SHINE II proposal is to identify cis-regulatory elements in SF3B1 intron 4 and SLC25A28 intron 2 that mediate IR, and to target key regulatory elements with antisense oligonucleotides in an effort to modulate IR efficiency. Regulatory elements will be studied in the context of newly constructed minigene splicing reporters already demonstrated to successfully model IR in transfected cells. Systematic mutation and deletion analysis of the splicing reporter will reveal which regions impact IR, and guide attempts to block these regions with antisense reagents in order to modulate IR. These studies are entirely novel since nothing is known about regulation of IR. The mechanistic information gained here will be relevant to many other genes that also execute IR as part of their expression repertoire, especially other RNA splicing factors that play a huge role in fine tuning transcriptome structure. In the future this data may lead to treatment strategies for emerging diseases characterized by intron retention.
Intron retention is a newly recognized component of the erythroid RNA processing program that is essential for normal differentiation and conversely, defective RNA processing underlies numerous human diseases. During terminal erythropoiesis, contrasting patterns of intron retention are observed in SF3B1, a critical RNA processing gene that is associated with a subtype of myelodysplasia, and in SLC25A28, a mitochondrial iron importer that impacts iron homeostasis. This project will study the molecular mechanism of intron retention in these transcripts, in order to gain therapeutic insights into ?intron retention diseases? we anticipate will be appreciated in the near future.
| Parra, Marilyn; Booth, Ben W; Weiszmann, Richard et al. (2018) An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys. RNA 24:1255-1265 |
| Conboy, John G (2017) RNA splicing during terminal erythropoiesis. Curr Opin Hematol 24:215-221 |
