ALBERT

Description: 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.

Description: Many protein factors that guide pre-mRNA modification pathways are composed of RNA recognition motif (RRM) domains. In an effort to identify splicing regulators in erythroid cells, we cloned a RRM-containing protein RBM25 and characterized its role in alternative splicing. RBM25 consists of a proline-rich region and a RRM domain at the amino-terminal end, an ER rich domain at the central region, and a PWI domain at the carboxyl terminal end. RBM25 partially co-localized with splicing factor SC35 in nuclear speckles. While both the RRM and PWI domains are diffusely distributed in the nucleoplasm, the ER domain is highly concentrated in nuclear speckles. We examined cellular mRNA targets of RBM25 in HeLa cells and demonstrated that it binds Bcl-x mRNA and affects its alternative splicing. Depletion of RBM25 by RNA interference caused accumulation of anti-apoptotic Bcl-x(L), whereas its up-regulation increased the levels of pro-apoptotic Bcl-x(s). The expression level of RBM25 also correlated with the degree of cell death, further suggesting that it functions in regulating apoptotic factor(s) expression. Apoptosis plays an important role in red cell development; earlier erythroid progenitors are more sensitive to apoptosis while mature erythroblasts are resistant to apoptosis. A significant decrease in RBM25 expression occurs during erythroid differentiation and correlates with resistance to apoptosis. Our results suggest that erythroblasts may acquire resistance to apoptosis during maturation through differential expression of crucial splicing regulators of the apoptotic machinery.

Description: Protein 4.1R is a vital component of the red blood cell membrane cytoskeleton. Promotion of cytoskeletal junctional complex stability requires an erythroid differentiation stage–specific splicing switch promoting inclusion of exon 16 within the spectrin/actin binding domain. We showed earlier that an intricate combination of positive and negative RNA elements controls exon 16 splicing. In this report, we further identified 3 putative exonic splicing enhancers within exon 16 and investigated the function of the sequence CAGACAT in the regulation of exon 16 splicing. Mutation of these sequences leads to increased exclusion of exon 16 in both in vivo and in vitro splicing assays, indicating that CAGACAT is a functional exonic splicing enhancer. UV cross-linking further detects an approximately 33-kDa protein that specifically binds to the CAGACAT-containing transcript. An anti-SF2/ASF antibody specifically immunoprecipitates the approximately 33-kDa protein. Furthermore, SF2/ASF stimulates exon 16 inclusion in both in vitro complementation assays and minigene-transfected mouse erythroleukemia cells (MELCs). Finally, SF2/ASF expression is up-regulated and correlates with exon 16 inclusion in differentiated MELCs. These results suggest that increased splicing factor 2/alternative splicing factor (SF2/ASF) expression in differentiated mouse erythroleukemia mediates a differentiation stage–specific exon 16 splicing switch through its interaction with the exonic splicing enhancer.

Description: Alternative splicing events altering the 5′ end of the red blood cell protein 4.1 (4.1R) mRNA are critical for modulating the expression of both the 135 kD and 80 kD isoforms. Three mutually exclusive 5′ exons, 1A, 1B, and 1C, are transcribed from their respective promoter and exhibit differential splicing to exon 2′/2 splice acceptor sites. Exon 1A splices to the distal 3′ splice site (3′ ss) excluding exon 2′ and encoding for the 80 kD isoform. Exons 1B and 1C splice to the proximal 3′ ss, including exon 2′ in mature mRNA and encoding for the 135 kD form. We investigated the regulatory mechanism involved in the selection of the alternative 3′ ss, using 1A, 1B, and 1C minigene constructs containing intronic sequences downstream of each exon joined with intronic sequences upstream of exon 2′. We positioned either CMV or the respective native promoter upstream of the minigene and analyzed the spliced products for exon 2′ expression in mouse (MELC or C2C12) or human (HeLa or RD) cells. When under the control of the CMV promoter, all 1A, 1B, and 1C minigenes resulted in the inclusion of exon 2′. However, when under the control of its respective promoter, the minigene mimicked its endogenous splicing pattern, suggesting that promoter identity influences the alternative exon 2′ splicing decision. To confirm this hypothesis, we switched 1A and 1B promoters in their respective minigene constructs. Replacement of the 1A promoter by the 1B promoter resulted in the inclusion of exon 2′ in the 1A minigene. Conversely, replacement of the 1B promoter by the 1A promoter caused increased exclusion of exon 2′ in the 1B minigene, confirming that alternative splicing of exon 2′ is sensitive to the type of promoter. The current model on the modulation of alternative splicing by promoters suggests that the promoter might control alternative splicing via the regulation of polymerase II (pol II) elongation or processivity. To test whether the same mechanism applies to exon 2′/2 splicing, we treated 1A and 1B minigene-transfected cells with transcription elongation inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). Inhibition of transcription elongation did not affect splicing of the 1B minigene, but it enhanced exon 2′ inclusion in the 1A minigene. The distal 3′ ss is a stronger splice site than the proximal 3′ ss. A highly processive elongating pol II favors the simultaneous presentation of both sites to the splicing machinery, a situation in which the distal 3′ ss out-competes the proximal 3′ ss, resulting in exon 2′ exclusion. Conversely, a slow pol II processivity on the 1B promoter favors the selection of the proximal 3′ ss and inclusion of exon 2′. Taken together, our results show that the alternative splicing of exon 2′/2 is tightly coupled to promoter architecture. Inclusion or exclusion of exon 2′ is achieved through a coordinated action of transcription and splicing.

Description: A regulated splicing event in protein 4.1R pre-mRNA—the inclusion of exon 16–encoding peptides for spectrin-actin binding—occurs in late erythroid differentiation. We defined the functional significance of an intronic splicing enhancer, UGCAUG, and its cognate splicing factor, mFox2A, on exon 16 splicing during differentiation. UGCAUG displays cell-type–specific splicing regulation in a test neutral reporter and has a dose-dependent enhancing effect. Erythroid cells express 2 UGCAUG-binding mFox-2 isoforms, an erythroid differentiation–inducible mFox-2A and a commonly expressed mFox-2F. When overexpressed, both enhanced internal exon splicing in an UGCAUG-dependent manner, with mFox-2A exerting a much stronger effect than mFox-2F. A significant reciprocal increase in mFox-2A and decrease in mFox-2F occurred during erythroid differentiation and correlated with exon 16 inclusion. Furthermore, isoform-specific expression reduction reversed mFox-2A–enhancing activity, but not that of mFox-2F on exon 16 inclusion. Our results suggest that an erythroid differentiation–inducible mFox-2A isoform is a critical regulator of the differentiation-specific exon 16 splicing switch, and that its up-regulation in late erythroid differentiation is vital for exon 16 splicing.

Description: The inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine erythroleukemia cells that reproduces the endogenous exon 16 splicing patterns from a transfected minigene. Exon 16 was excluded in predifferentiated and predominantly included after induction. This suggests that the minigene contained exon and abutting intronic sequences sufficient for splicing regulation. A systematic analysis of the cis-acting regulatory sequences that reside within the exon and flanking introns was performed. Results showed that (1) the upstream intron of 4.1R pre-mRNA is required for exon recognition and it displays 2 enhancer elements, a distal element acting in differentiating cells and a proximal constitutive enhancer that resides within the 25 nucleotides preceding the acceptor site; (2) the exon itself contains a strong constitutive splicing silencer; (3) the exon has a weak 5′ splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating cells, a proximal element at the vicinity of the 5′ splice site, and a distal element containing 3 copies of the UGCAUG motif. These results suggest that the interplay between negative and positive elements may determine the inclusion or exclusion of exon 16. The activation of the enhancer elements in late erythroid differentiation may play an important role in the retention of exon 16.

Description: Protein 4.1R (4.1R), a vital component of the red cell membrane cytoskeleton, stabilizes the spectrin-actin lattice and attaches it to embedded membrane proteins. A regulated splicing event, the inclusion of exon 16 that encodes for peptides critical for spectrin/actin binding, occurs during late erythroid differentiation. We showed earlier that an intricate combination of enhancer and silencer elements direct exon 16 splicing. Regulated expression of splicing factors, SF2/ASF and hnRNP A/B, has also been implicated in mediating exon 16 splicing. In this study, we attempted to characterize the mechanism involved in exon 16 splicing through UGCAUG, an intronic splicing enhancer present in three copies in the intron downstream of exon 16. We first used a wild-type minigene construct consisting of exons 13, 16, 17 and their respective flanking introns that mimics endogenous exon 16 splicing during the induced differentiation of mouse erythroleukemia cells (MELC). Mutational analysis showed a dose-dependent effect of UGCAUG on exon 16 splicing: the presence of all three copies had the most effect. These results were recapitulated with an internal chimeric exon in a test neutral reporter system “DUP4-1”, suggesting that the enhancing effect could be attributed directly to UGCAUG. Furthermore, we identified a novel splicing factor, RBM-9, from MELC that enhanced the internal exon splicing in an UGCAUG-dependent manner in both the exon 16 minigene and DUP4-1 reporter systems. Our characterization of RBM-9 revealed that diverse isoforms of RBM-9 are generated by the utilization of alternative translation initiation sites and tissue-specific alternative splicing; different isoforms from various tissues exhibited differential exon 16 splicing enhancing activities. MELC-RBM-9 enhanced exon 16 splicing the most among all RBM-9 isoforms tested. Inhibition of RBM-9 expression by RBM-9-shRNA reversed its enhancing activity on exon 16 inclusion in MELC. RBM-9-shRNA also reduced exon 16 splicing in a dose-dependent manner in HeLa cells. Furthermore, purified RBM-9 specifically binds to the UGCAUG sequence in a gel-mobility shift assay. Finally, expression of RBM-9 is upregulated and correlates with exon 16 inclusion during MELC differentiation. These results suggest that a novel splicing factor, RBM-9, enhances erythroid differentiation stage-specific exon 16 splicing by interacting with the splicing enhancer UGCAUG.

Description: Abstract 778 Erythroid differentiation-induced Fox-2A is an important regulator for the differentiation-specific exon 16 splicing switch in protein 4.1R. Up-regulation of Fox-2A in late erythroid differentiation is critical for inclusion of exon 16, which encodes a portion of the spectrin/actin binding domain vital for maintaining the mechanical stability of red blood cell membranes. Fox-2A exerts its splicing enhancing activity in a motif-dependent manner, binding to UGCAUGs located downstream of exon 16. In this study, we investigated the mechanism by which Fox-2A modulates the expression of exon 16. The excision of introns and the joining of exons depends on the recognition and usage of 5' and 3' splice sites (5' ss and 3' ss, respectively) by the splicing machinery. Exon 16 possesses a relatively strong 3' ss but a weak 5' ss. Mutation of the weak 5' ss (GAGIGTTTGT) to a strong consensus 5' ss (GAGIGTAAGT) led to nearly total exon 16 inclusion. While mutations impairing Fox-2A binding drastically reduced exon 16 inclusion in the presence of the weak 5' ss, no effect on exon 16 inclusion was observed when the strong 5' ss was presented with these mutations. These results suggest that Fox-2A facilitates exon 16 splicing by supporting the weak 5' ss. Early recognition of the 5' ss involves base-pairing interaction with the 5' end of U1 snRNA and stabilization by U1 snRNP. Psoralen-mediated UV cross-linking assays revealed a reduction in U1 snRNA recruitment to the weak 5' ss when Fox-2A binding sites were impaired, suggesting that binding of Fox-2A could promote recruitment and stabilization of U1 snRNP to the weak 5' ss. In support for a role of Fox-2A in modulating the activation of the weak 5' ss by recruiting U1 snRNP, we demonstrated that Fox-2A directly interacts with U1 specific protein U1C in an RNA-independent manner. The C-terminal domain of Fox-2A is responsible for its association with U1C. These data suggest a novel mode for exon 16 5' ss activation in which the binding of Fox-2A to an intronic splicing enhancer element UGCAUG may stabilize the pre-mRNA-U1 snRNP complex through interactions with U1C. These could then result in spliceosome commitment complex formation and exon 16 inclusion. Disclosures: No relevant conflicts of interest to declare.