Alternative pre-mRNA splicing is one of the central mechanisms for the regulation of gene expression in eukaryotic cells. It allows the generation of functionally distinct proteins from a single gene. It has been estimated that 40–60% of human genes are alternatively spliced. Moreover, alternative splicing is often regulated in a cell-type, tissue or developmentally specific manner [for reviews, see (1–3
The splicing reaction is carried out by the spliceosome, a large ribonucleoprotein complex containing five small nuclear ribonucleoproteins (snRNPs) and many protein splicing factors. Spliceosome assembly occurs in an ordered manner within each intron. The initial step for spliceosome formation is assembly of early (E) complex (4
): U1 snRNP interacts with the 5′ splice site, SF1 (splicing factor 1) binds to the branch point, and the U2AF65/35 heterodimer binds to the pyrimidine tract and the 3′ splice site. In an ATP requiring step, U2 snRNP tightly associates with the branch site, generating the A complex. Subsequently, the U4/U6/U5 tri-snRNPs associate to the A complex to form the B complex. After RNA–RNA rearrangements occur, the catalytically activated spliceosome is formed. During these rearrangements, the U1 and U4 snRNPs dissociate and the U6 snRNA contacts with the 5′ splice site and U2 snRNA. This is the catalytic C complex spliceosome in which the two trans
-esterification reactions of splicing occur, resulting in exon ligation and lariat intron release (6–8
Spliceosome assembly is regulated by several non-spliceosomal RNA-binding proteins, such as SR and hnRNP proteins. SR proteins usually play key roles in constitutive and alternative splicing, by mediating splicing activation via binding to exonic splicing enhancers (ESEs). In contrast, hnRNP proteins act as splicing repressors via binding to exonic splicing silencers (ESSs) and intronic splicing silencers (ISSs) (9
). These proteins are extensively studied for their effect to spliceosome assembly in alternative splicing, and are thought to affect the initial step of spliceosome assembly, the E complex formation.
Recently, several tissue-specific splicing regulators have been reported. For example, a neuron-specific RNA-binding protein, Nova-1, binds to the RNA sequence UCAUY and regulates the alternative splicing of several genes such as glycine receptor a2 (10
). The CELF (CUG-BP and ETR3-like factors) family proteins are implicated in regulation of tissue-specific splicing of several genes, including cTNT, IR and α-actinin (12–14
In our previous study, we identified vertebrate homologs of the Caenorhabditis elegans
Fox-1 protein in zebrafish and mouse. Fox-1 is an RNA-binding protein that contains an RNA recognition motif (RRM). In mouse, Fox-1 is expressed in brain, heart and skeletal muscle. Our SELEX experiments showed that zebrafish Fox-1 protein binds specifically to the pentanucleotide GCAUG (15
). Interestingly, it has been reported that (U)GCAUG is essential for the alternative splicing of several genes (3
). Furthermore, a recent computational analysis revealed that the UGCAUG element is overrepresented in the downstream introns of neuron-specific exons and is conserved among vertebrate species (16
Fox-1 induces muscle-specific exon skipping through binding to the GCAUG repressor element upstream of alternative exon in the human mitochondrial ATP synthase γ subunit (hF1γ) gene (15
). In the case of calcitonin/CGRP, two copies of UGCAUG in the upstream intron and the regulated exon are essential for the induction of exon skipping by Fox-1 or its paralog Fox-2 (17
). In contrast, exon inclusion in fibronectin, non-muscle myosin heavy chain (NMHC)-B, c-src and FGFR2, 4.1R is induced by Fox proteins via the (U)GCAUG enhancer element in the downstream intron (15
). Thus, in the known cases so far, the (U)GCAUG element that resides in the intron upstream of alternative exon functions as a repressor element, whereas the element that activates exon inclusion is found in the intron downstream of the alternative exon. Thus, it is likely that Fox proteins function as both splicing repressor and activator, depending on where they bind relative to the affected exon. However, little is known about the molecular mechanisms of how Fox proteins regulate such alternative splicing.
To examine the molecular mechanism of exon skipping by Fox-1, we studied its effect on the spliceosome assembly using the hF1γ gene as a model. Here we report that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor element in intron 8. The splicing efficiency of intron 8 was not affected much by Fox-1 protein. In vitro splicing analyses show that Fox-1, by binding to the GCAUG element in intron 8, prevents formation of the pre-spliceosomal E complex onto intron 9. Such repression by Fox-1 represents a novel mechanism for splicing regulation by tissue-specific splicing regulators. In addition, we identified a region of the Fox-1 protein that is required for inducing the exon skipping, suggesting that this region plays a key role in interacting with other splicing factor(s) to regulate alternative splicing.