The Fox-1 family of RNA-binding proteins are evolutionarily conserved regulators of tissue-specific alternative splicing in metazoans. The Fox-1 family specifically recognizes the (U)GCAUG stretch in regulated exons or in flanking introns, and either promotes or represses target exons. Recent unbiased bioinformatics analyses of alternatively spliced exons and comparison of various vertebrate genomes identified the (U)GCAUG stretch as a highly conserved and widely distributed element enriched in intronic regions surrounding exons with altered inclusion in muscle, heart, and brain, consistent with specific expression of Fox-1 and Fox-2 in these tissues. Global identification of Fox-2 target RNAs in living cells revealed that many of the Fox-2 target genes themselves encode splicing regulators. Further systematic elucidation of target genes of the Fox-1 family and other splicing regulators in various tissues will lead to a comprehensive understanding of splicing regulatory networks.

Many pre-mRNAs are alternatively spliced in a tissue-specific manner in multicellular organisms. The Fox-1 family of RNA-binding proteins regulate alternative splicing by either activating or repressing exon inclusion through specific binding to UGCAUG stretches. However, the precise cellular contexts that determine the action of the Fox-1 family in vivo remain to be elucidated. We have recently demonstrated that ASD-1 and FOX-1, members of the Fox-1 family in Caenorhabditis elegans, regulate tissue-specific alternative splicing of the fibroblast growth factor receptor gene, egl-15, which eventually determines the ligand specificity of the receptor in vivo. Here we report that another RNA-binding protein, SUP-12, coregulates the egl-15 alternative splicing. By screening for mutants defective in the muscle-specific expression of our alternative splicing reporter, we identified the muscle-specific RNA-binding protein SUP-12. We identified juxtaposed conserved stretches as the cis elements responsible for the regulation. The Fox-1 family and the SUP-12 proteins form a stable complex with egl-15 RNA, depending on the cis elements. Furthermore, the asd-1; sup-12 double mutant is defective in sex myoblast migration, phenocopying the isoform-specific egl-15(5A) mutant. These results establish an in vivo model that coordination of the two families of RNA-binding proteins regulates tissue-specific alternative splicing of a specific target gene.

RBM20 is a vertebrate-specific RNA-binding protein with two zinc finger (ZnF) domains, one RNA-recognition motif (RRM)-type RNA-binding domain and an arginine/serine (RS)-rich region. RBM20 has initially been identified as one of dilated cardiomyopathy (DCM)-linked genes. RBM20 is a regulator of heart-specific alternative splicing and Rbm20^ΔRRM mice lacking the RRM domain are defective in the splicing regulation. The Rbm20^ΔRRM mice, however, do not exhibit a characteristic DCM-like phenotype such as dilatation of left ventricles or systolic dysfunction. Considering that most of the RBM20 mutations identified in familial DCM cases were heterozygous missense mutations in an arginine-serine-arginine-serine-proline (RSRSP) stretch whose phosphorylation is crucial for nuclear localization of RBM20, characterization of a knock-in animal model is awaited. One of the major targets for RBM20 is the TTN gene, which is comprised of the largest number of exons in mammals. Alternative splicing of the TTN gene is exceptionally complicated and RBM20 represses >160 of its consecutive exons, yet detailed mechanisms for such extraordinary regulation are to be elucidated. The TTN gene encodes the largest known protein titin, a multi-functional sarcomeric structural protein specific to striated muscles. As titin is the most important factor for passive tension of cardiomyocytes, extensive heart-specific and developmentally regulated alternative splicing of the TTN pre-mRNA by RBM20 plays a critical role in passive stiffness and diastolic function of the heart. In disease models with diastolic dysfunctions, the phenotypes were rescued by increasing titin compliance through manipulation of the Ttn pre-mRNA splicing, raising RBM20 as a potential therapeutic target.

Alternative splicing of pre-mRNAs greatly contributes to the spatiotemporal diversity of gene expression in metazoans. However, the molecular basis of developmental regulation and the precise sequence of alternative pre-mRNA processing in vivo are poorly understood. In the present study, we focus on the developmental switching of the mutually exclusive alternative splicing of the let-2 gene of Caenorhabditis elegans from the exon 9 form in embryos to the exon 10 form in adults. By visualizing the usage of the let-2 mutually exclusive exons through differential expression of green fluorescent protein (GFP) and red fluorescent protein (RFP), we isolated several switching-defective mutants and identified the alternative splicing defective-2 (asd-2) gene, encoding a novel member of the evolutionarily conserved STAR (signal transduction activators of RNA) family of RNA-binding proteins. Comparison of the amounts of partially spliced let-2 RNAs in synchronized wild-type and asd-2 mutant worms suggested that either of the introns downstream from the let-2 mutually exclusive exons is removed prior to the removal of the upstream ones, and that asd-2 promotes biased excision of intron 10 in the late larval stages. We propose that the developmental switching between alternative sequences of intron removal determines the ratio between the mature let-2 mRNA isoforms.