Many neuronally regulated exons are controlled by intronic splicing enhancers containing the element UGCAUG (4
). We show that mammalian Fox-1 and its homologue Fox-2 are specifically expressed in neurons and that both proteins activate neuron-specific exons through UGCAUG-dependent enhancers. Recently, it was demonstrated that Fox proteins could also enhance splicing of a neuron-specific exon from the nonmuscle myosin heavy chain pre-mRNA (39
). Thus, the Fox proteins are likely key regulators of splicing in the nervous system.
In addition to neurons, the Fox proteins also play roles in other tissues. Fibronectin exon EIIIB, whose inclusion is not neuron specific, is also dependent upon Fox proteins. The proteins are also expressed in heart and muscle (22
). In muscle cells, Fox-1 was shown to act as a splicing repressor, inhibiting the splicing of exons in actinin and ATP synthase transcripts. These exons are normally skipped in muscle but used elsewhere (Fig. ). Interestingly, the GCAUG regulatory elements needed for this repression are upstream of the skipped exons. So far, all of the UGCAUG enhancer elements known to activate splicing are found downstream of the regulated exon (36
) (Fig. ). Thus, the Fox proteins can apparently act either positively or negatively, depending on where they bind relative to the affected exon. Similar location dependence is seen in the SR proteins that activate splicing when bound in exons but inhibit splicing when bound to intronic elements (14
FIG. 8. Fox proteins bind to introns to influence the splicing of adjacent exons by multiple mechanisms. (A) Fox promotes exon skipping. In muscle cells, Fox proteins bind upstream of a non-muscle-specific exon (NM) to promote skipping (22). (B) Fox promotes (more ...)
In addition to the location of the Fox protein binding site, the particular Fox isoform expressed is likely to affect its activity. In mammals, Fox-1 and Fox-2 are members of an extended family of proteins arising from multiple genes and complex alternative splicing. Recent work demonstrated that the expression of certain spliced isoforms is specific to particular tissues and that their enhancer activities are variable (39
). It will thus be important to characterize which forms are expressed in which cell types and to determine whether particular isoforms are needed for the regulation of specific transcripts.
The Fox proteins have been identified in other contexts. In C. elegans
, the original Fox gene is required for proper assessment of the X-to-autosome ratio for dosage compensation during sexual development (19
). Interestingly, this worm protein controls a downstream transcript, Xol-1, which exhibits alternative splicing patterns. This worm system may show an effect on splicing different from that seen in mammals. The sixth intron of Xol-1 is more efficiently spliced in the absence of Fox-1, which may cause its retention (Fig. ) (43
). This intron contains two copies of the pentamer GCAUG, but whether this is the target sequence for the worm protein has not been reported. The RRM of the worm Fox protein is 75% identical to the mouse homologues, and its auxiliary domains have similarities to mammalian splice variants. Worm Fox-1 also functions in the adult, where it is expressed in some neurons and other cells. Thus, Fox-1 may function in the adult worm in a manner similar to its role in mammalian cells. Conversely, there may be additional embryonic functions for the mammalian protein.
The mammalian Fox proteins have also been identified in other studies. Fox-1 was identified in yeast two-hybrid screens as interacting with ataxin-2 and was called A2bp1 (42
). Trinucleotide expansions in the ataxin-2 gene cause spinocerebellar ataxia type 2 (21
). Interestingly, ataxin-2 also has homologues in worms and other distantly related species and is also thought to be an RNA binding protein (10
). A2bp1 has been reported to localize to the Golgi apparatus (26
). We see predominantly the nuclear localization expected for a splicing regulator. However, we cannot rule out the possibility that the cytoplasmic population is Golgi associated. It also appears that different isoforms of the protein show differential localization (39
Interestingly, human mutations in A2bp1 were identified in a limited number of patients with an inherited epilepsy and mental retardation disorder (3
). The Fox1/A2bp1 gene also lies within an autism susceptibility locus on chromosome 16 (2
). Given its function described here, these mutations in Fox-1 will lead to changes in the neuronal regulation of splicing. Thus, it will be very interesting to test for changes in the splicing of UGCAUG-dependent exons in multiple forms of neurological disease, including ataxias, epilepsy, and autism. The recent implication of splicing defects in other neurological disorders, such as frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) and myotonic dystrophy, provides an interesting precedent for this idea (15
Studies of Fox-2 (RBM9) identified it as a gene upregulated by androgens and as a repressor of tamoxifen activation of the estrogen receptor (28
). There are splice variants of Fox-2 that, judging from EST frequency data, appear specific to breast, ovary, and other estrogen-sensitive tissues (data not shown). Thus, hormone signaling may regulate alternative splicing through changes in Fox transcription or isoform ratios. Analysis of downstream Fox targets in these tissues is likely to uncover another important role for Fox proteins.
The mechanism of splicing activation by the Fox proteins and their interaction with other splicing regulators are also interesting directions for future studies. Fox-regulated exons often show repression by PTB, and PTB binding elements are often found adjacent to Fox sites. It will be interesting to examine whether these proteins bind in a common regulatory complex or perhaps antagonize each other's binding.
The roles of the conserved N- and C-terminal Fox domains are also unknown. Understanding their interactions with the general splicing machinery will be important in determining the mechanism of splicing regulation. Most intronic regulatory elements studied to date cluster near 5′ or 3′ splice sites and often overlap with them (5
). The neuronal exons studied here have a UGCAUG element within 100 nucleotides of the alternative 5′ splice site. Many other neuron-specific exons have at least one conserved UGCAUG in this region (6
). However, fibronectin exon EIIIB and calcitonin/CGRP carry important UGCAUG elements at a distance of several hundred nucleotides from the affected splice sites (18
). Splice site proximity and enhancer location relative to the exon are additional features that must be understood to predict Fox-regulated exons and whether Fox will be a repressor or enhancer.
UGCAUG is among the most precisely conserved splicing regulatory elements. The presence of these elements in groups of commonly regulated exons is a clue to the biological role of regulation by Fox proteins (36
). This biological role is likely conserved across diverse species. Genetic analyses in the worm, fish, and mouse will yield information on their common function in the lives of these different organisms.