Protein 4.1R Exon 16 Splicing Mediated by Regulated Expression of RBM9 and PTB during Erythroid Differentiation.

Protein 4.1R (4.1R), a vital component of the red cell membrane cytoskeleton, stabilizes the spectrin-actin lattice and attaches it to the embedded membrane proteins. The inclusion of exon 16, which encodes peptides critical for spectrin/actin binding, occurs via an intricate interplay between the auxiliary cis-elements and transacting factors. An intronic splicing enhancer, UGCAUG, is present in triplicate and is situated between two polypyrimidinetract-binding (PTB) sites, TCTT, in the intron downstream of exon 16. In addition, PTB binding sites are also present in triplicate in the upstream intron of exon 16. In this study, we characterized the splicing factors that orchestrate the erythroid differentiation stage-specific switch in exon 16 splicing through these cis-elements using two cell systems: mouse erythroleukemia cells (MELC) that can be induced to erythroid differentiation and G1E-ER cells that undergo synchronous erythroid maturation after induced GATA-1 expression. We identified two RBM9 isoforms (RBM9-1A and RBM9-1F) with distinct amino-termini that interact with the intronic splicing enhancer UGCAUG. The expression of RBM9-1A is erythroid-specific while RBM9-1F can be detected in a wide variety of cell types. Real-time PCR and Western blot analyses showed that RBM9-1A expression is significantly increased while RBM9-1F is reduced during induced erythroid differentiation in both MELC and G1E-ER4 cells. The up-regulation of RBM9-1A correlated with exon 16 inclusion in differentiated cells. Furthermore, the inhibition of RBM9 expression by isoform specific-shRNA reversed 1A enhancing activity, but not that of 1F on exon 16 inclusion in differentiated cells. Thus, exon 16 splicing is mediated by a cell type-specific RBM9 isoform and its up-regulation in late erythroid differentiation is vital for exon 16 splicing. However, over-expression of PTB completely diminished the enhancing effect of RBM9-1A on exon 16 splicing in both differentiated MELC and G1E-ER4 cells, suggesting that PTB plays a role in exon 16 splicing. We analyzed PTB expression and its effect on the exon 16 splicing switch during erythroid differentiation. PTB, a repressive regulator of alternative splicing, binds to the exon 16 upstream and downstream intronic silencers. Its over-expression reduced exon 16 inclusion in both endogenous 4.1R and transfected exon 16 minigenes. Moreover, PTB expression was down-regulated and coincided with increased exon 16 splicing during erythroid differentiation suggesting that regulated expression of repressor PTB mediates exon 16 splicing. Our results further suggest that the differentiation-specific exon 16 splicing switch is achieved by varying the amount of either ubiquitously expressed or cell-type specific activators and inhibitors, and hence the relative efficiency of spliceosome recruitment in the exon inclusion pathway.