An Intron Splicing Enhancer Element, UGCAUG, Is Evolutionarily Conserved near Erythroid Protein 4.1R Exon 16 and Other Tissue-Specific Alternative Exons.
Authors: Gee Sherry L. , Lersch Robert L. , Minovitsky Simon , Ponthier Julie L. , Lo Annie J. , Short Sarah , Chasis Joel A. , Dubchak Inna , Conboy John G. 
Published: October 2019

Abstract
Alternative pre-mRNA splicing switches provide an important mechanism for regulating gene expression during differentiation. In differentiating erythroblasts, stage-specific activation of protein 4.1R exon 16 splicing promotes the synthesis of protein isoforms with high affinity for spectrin and actin, which is important for mechanical stabilization of the red cell membrane skeleton. Regulation of this alternative splicing switch is mediated by the interaction of multiple trans-acting splicing factor proteins with cis RNA elements in the pre-mRNA. We previously identified an intron splicing enhancer downstream of exon 16 that is essential for optimal inclusion of exon 16 in functional splicing assays. This enhancer contains three repeats of the sequence UGCAUG, a highly specific binding site for the newly described Fox-1 family of splicing activator proteins. Our results implicate the highly homologous Fox-2 protein as a candidate splicing activator for exon 16 inclusion during erythroid differentiation, based on the observations that Fox-2 is expressed in mouse erythroblasts; and that it both binds to the intron 16 enhancer, and enhances exon 16 splicing, in a UGCAUG-dependent manner. Here we also report genome sequence analyses showing that UGCAUG is evolutionarily conserved in the proximal downstream intron sequence near many tissue-specific alternative exons. First, examination of the 4.1R genes revealed that three repeats of the UGCAUG motif were conserved in introns of all mammals tested (human, chimp, mouse, rat and dog) as well as in chicken; two repeats were found in intron 16 of the frog; and one repeat in the zebrafish. These data suggest that Fox-2 may be important for regulating exon 16 splicing from fish to man. Next, to test whether a similar mechanism might regulate other tissue-specific exons, we analyzed a larger group of some 27 alternative exons with predominant expression in the brain. Remarkably, 80–90% of these exons possessed one or more UGCAUG motifs within 1kb of flanking intron sequence. The majority of these elements were concentrated in the intron 10–400nt downstream of the regulated exons. Phylogenetic analysis of individual exons revealed that the number and position of intronic UGCAUG elements were highly conserved among mammalian species and in the chicken, but more divergent in fish. In marked contrast, control datasets of constitutively spliced exons, and alternatively spliced exons not known to be regulated in tissue-specific patterns, exhibited a low incidence of UGCAUG elements in the flanking introns. These findings are exactly as expected for a functionally important regulatory element. We propose that the high binding specificity of Fox proteins, and the unique localization of UGCAUG enhancers near regulated alternative exons, is advantageous for splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in specific cell types including erythroblasts. We speculate that erythroid-specific splicing is accomplished by coordination between Fox-2 and other as yet unknown splicing factors.