Regulation of Exon 2′/2 of Protein 4.1R by Coordinated Transcription and Splicing.

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.