Previous studies have shown that UGCAUG is an intronic splicing regulatory element (18
), that it is bound with unusually high specificity by the novel alternative splicing factor, Fox-1 (35
), and that it is required for efficient splicing of neural-specific alternative exons (18
). The demonstration that UGCAUG expression is widely associated with brain-enriched exons (15
) and is evolutionarily conserved from fish to man (this paper) further implicates the Fox-UGCAUG system as an important regulator of tissue-specific alternative splicing. Taken together, these biochemical and computational findings suggest a model whereby Fox protein binding to UGCAUG plays a critical role in activating splicing switches for many alternative exons during neural development and differentiation.
As shown in this paper, the intronic UGCAUG motif is phylogenetically and spatially conserved near brain-enriched exons. The majority of these brain-enriched exons possess at least one copy of UGCAUG in the flanking intron, and the remaining few generally have closely related GCAUG pentameric motif(s) in the proximal intron [(15
); and from results not shown]. Evolutionary conservation of intronic UGCAUG elements has been noted previously (27
). However, biochemical studies have demonstrated that in some cases the GCAUG pentamer is functional in splicing assays (25
). We propose that the presence of linked (U)GCAUG elements may be an essential property for a specialized subset of alternative exons whose splicing is both tissue-specifically regulated and phylogenetically conserved. Consistent with this model, our studies did not identify UGCAUG in association with either constitutive exons or non-tissue-specific alternative exons. We speculate that other experimental approaches such as RESCUE-ISE (13
) and the use of support vector machines (45
) have not yet identified UGCAUG as a candidate regulatory element only because they have not been applied to the analysis of tissue-specific alternative exons. Almost certainly, these powerful techniques will be invaluable for future studies of splicing regulatory elements.
Alternative exons typically exhibit higher phylogenetic conservation of proximal intron sequences than do constitutive exons (43
). Presumably, this conservation reflects a functional role in regulation of alternative splicing; our analyses begin to reveal specific functional motifs characteristic of these conserved introns. Besides the over-representation of UGCAUG elements, introns flanking alternative exons are modestly deficient in purines in general, and highly deficient in G-triplets in particular, compared with the introns flanking control exons. This was a robust finding in all six species-specific brain datasets examined in the study. Since G-triplets have been shown to enhance splicing of neighboring exons (39
), it is tempting to speculate that the absence of this widely expressed class of intronic enhancers may contribute to the default skipping phenotype of many alternative exons. The ability to efficiently activate splicing of these exons might then require another class of intronic enhancer, e.g. the UGCAUG element or analogous motifs, in order to achieve tissue-specific regulation during development and differentiation. Alternative exons that lack both G-triplets and UGCAUG element(s) might represent a separate class that does not exhibit tissue-specific switching.
We propose that the functional importance of the UGCAUG motif is to serve as a critical cis
-acting component of alternative splicing switches that trigger many developmental- and differentiation-specific changes in pre-mRNA splicing. Furthermore, we propose that Fox-1 related proteins represent the core of the conserved regulatory machinery that mediates these splicing switches. Direct experimental evidence for Fox-1 splicing regulation via (U)GCAUG motif(s) has been reported (35
). Moreover, the RRM domains of human A2BP1/Fox-1 (46
) and zebrafish Fox-1 (35
) are highly conserved (~91% identity), and comparative genomic analysis indicates that this domain in the mouse, dog, rat and chicken orthologs is identical to human Fox-1 (our unpublished observations). Thus, the UGCAUG-Fox-1 system appears to have been highly conserved through evolution. The unique sequence specificity of this machinery may be an advantageous property for a splicing regulatory network designed to regulate a restricted population of exons in tissue-specific splicing patterns. However, a single isoform of Fox-1 alone clearly cannot account for the diversity of splicing patterns characteristic of complex genes in metazoan organisms. Indeed, the proteome's repertoire of Fox proteins may be fairly complex, involving multiple genes and multiple protein isoforms encoded by each gene. Future studies will be required to assess the relative RNA binding specificities and co-factor binding capabilities for each Fox isoform, in order to elucidate the rules that govern this regulatory system. Such studies may explain why the zebrafish Fox-1 preferentially binds the GCAUG pentamer in SELEX binding assays (35
), yet the UGCAUG hexamer exhibits preferential conservation in the genome. Moreover, it seems likely that a variety of splicing co-factors must cooperate with Fox proteins to activate splicing switches for highly selective groups of exons at the appropriate time and place during development. Such co-factors may include members of the SR and hnRNP families, factors related to NOVA-1 (47
) or CUG-binding proteins of the CELF family (22
), or proteins yet to be characterized. Future experiments will be aimed at identifying additional motifs and co-factors that cooperate with UGCAUG-Fox to determine precise timing of splicing switches in various cell types.