Mechanisms That Link Promoter Choice with Downstream Alternative Splicing in the Erythroid Protein 4.1R Gene.
Authors: Parra Marilyn , Tan Jeff , Mohandas Narla , Conboy John G. 
Published: October 2019

Abstract
The protein 4.1R gene is a large transcription unit (240kb) that utilizes complex RNA processing pathways to encode distinct protein isoforms, both during erythropoiesis and also in nonerythroid cells. Proper regulation of these pathways is essential for stage-specific synthesis of the 80-kDa isoforms of 4.1R protein during terminal erythroid differentiation. The 5′ region of the gene contains multiple alternative first exons that map far upstream of the coding exons, and we have shown previously that promoter choice is coupled to alternative splicing decisions 100kb downstream in exon 2′/2. Transcripts that initiate at exon 1A predominate in late stages of erythropoiesis and splice only to a weak internal 3′ splice acceptor site in exon 2, skipping translation start site AUG1 and ensuring proper translation initiation at AUG2 in exon 4 for synthesis of the 80-kDa isoforms. In contrast, 4.1 transcripts initiated at exons 1B or 1C exclusively splice to the strong first 3′ splice acceptor site at exon 2′ to include AUG1 and encode a higher molecular weight 135-kDa isoform known to interact with different affinity to major erythroid membrane proteins in earlier stages of erythropoiesis. Our studies show that this linkage between transcription and splicing is (a) cell type independent; (b) conserved in the 4.1R gene from fish to man; and (c) conserved in the paralogous 4.1B gene.
Our recent functional studies suggest that a novel re-splicing mechanism, reminiscent of recursive splicing of large introns previously described in the Drosophila ubx gene, may couple promoter choice with downstream splicing in the 4.1R gene. Using minigenes that reproduce the differential splicing patterns in transfected mammalian cells, we have shown that accurate splicing of exon 1A requires a unique downstream regulatory element. This element maps several kilobases downstream of exon 1A and is conserved among mammals. Analysis of wild type and mutated minigenes suggests a two step splicing model in which this element behaves as a temporary “intra-exon” that is present in a splicing intermediate but eliminated from the mature mRNA. According to this model, the regulatory element behaves as an exon in the first step as its consensus 5′ donor site splices to the strong 3′ splice site of exon 2′, removing this splice site pair and joining the intra-exon directly to exon 2′. In the second step, the juxtaposed region of the intra-exon then behaves as an intron, contributing to the activation of the weak internal splice acceptor at exon 2. This second splicing event joins exon 1A to exon 2, thus deleting the intra-exon, the 2′ region (and AUG1) and generating a mature 5′ end capable of encoding 80-kDa 4.1R. Importantly, pre-mRNA constructs that lack the intra-exon, or have a mutated intra-exon 5′ splice donor site, are uncoupled and exhibit inappropriate splicing of exon 1A to the first acceptor site at exon 2′. In support of the generality of this model, we have identified a candidate intra-exon with similar sequence properties in the long 5′ region of the human 4.1B gene, and have demonstrated that this element successfully rescues proper splicing of 4.1R exon 1A in our minigenes. Detailed molecular analysis is under way to identify the specific cis and trans elements required to effect this unusual, long-distance coupling between RNA processing events which have implications for detailed mechanistic understanding of membrane assembly during erythropoiesis.