Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1gamma gene

Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U)GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U)GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1γ gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.     

The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo

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.     

Muscle-specific exonic splicing silencer for exon exclusion in human ATP synthase gamma-subunit pre-mRNA.

Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.     

Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.     

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.     

Differential regulation of exonic regulatory elements for muscle-specific alternative splicing during myogenesis and cardiogenesis

Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pu-del and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.     

Repression of prespliceosome complex formation at two distinct steps by Fox-1/Fox-2 proteins

Precise and robust regulation of alternative splicing provides cells with an essential means of gene expression control. However, the mechanisms that ensure the tight control of tissue-specific alternative splicing are not well understood. It has been demonstrated that robust regulation often results from the contributions of multiple factors to one particular splicing pathway. We report here a novel strategy used by a single splicing regulator that blocks the formation of two distinct prespliceosome complexes to achieve efficient regulation. Fox-1/Fox-2 proteins, potent regulators of alternative splicing in the heart, skeletal muscle, and brain, repress calcitonin-specific splicing of the calcitonin/CGRP pre-mRNA. Using biochemical analysis, we found that Fox-1/Fox-2 proteins block prespliceosome complex formation at two distinct steps through binding to two functionally important UGCAUG elements. First, Fox-1/Fox-2 proteins bind to the intronic site to inhibit SF1-dependent E′ complex formation. Second, these proteins bind to the exonic site to block the transition of E′ complex that escaped the control of the intronic site to E complex. These studies provide evidence for the first example of regulated E′ complex formation. The two-step repression of presplicing complexes by a single regulator provides a powerful and accurate regulatory strategy.     

Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes

Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue-specific isoforms of a target gene that expedites a highly conserved signaling pathway.     

Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo

Alternative splicing of pre-mRNAs allows multicellular organisms to create a huge diversity of proteomes from a finite number of genes. But extensive studies in vitro or in cultured cells have not fully explained the regulation mechanisms of tissue-specific or developmentally regulated alternative splicing in living organisms. Here we report a transgenic reporter system that allows visualization of expression profiles of mutually exclusive exons in Caenorhabditis elegans. Reporters for egl-15 exons 5A and 5B showed tissue-specific profiles, and we isolated mutants defective in the tissue specificity. We identified alternative-splicing defective-1 (asd-1), encoding a new RNA-binding protein of the evolutionarily conserved Fox-1 family, as a regulator of the egl-15 reporter. Furthermore, an asd-1;fox-1 double mutant was defective in the expression of endogenous egl-15 (5A) and phenocopied egl-15 (5A) mutant. This transgenic reporter system can be a powerful experimental tool for the comprehensive study of expression profiles and regulation mechanisms of alternative splicing in metazoans.     

Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.     

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.     

Genome-wide detection of tissue-specific alternative splicing in the human transcriptome

We have developed an automated method for discovering tissue-specific regulation of alternative splicing through a genome-wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue-specific alternative splice forms of human genes. We validated our muscle-specific and brain-specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue-specific alternative splice forms is highest in brain, while eye-retina, muscle, skin, testis and lymph have the greatest enrichment of tissue-specific splicing. Overall, 10-30% of human alternatively spliced genes in our data show evidence of tissue-specific splice forms. Seventy-eight percent of our tissue-specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue-specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney-specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N-terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue-specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.     

Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle.

The alternative exon 5 of the striated muscle-specific cardiac troponin T (cTNT) gene is included in mRNA from embryonic skeletal and cardiac muscle and excluded in mRNA from the adult. The embryonic splicing pattern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonmuscle cell lines, minigenes express a default exon skipping pattern. Using this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-specific splicing. In this study, we identify four intron elements, one located upstream,and three located downstream of the alternative exon, which act in a positive manner to mediate the embryonic splicing pattern of exon inclusion. Synergistic interactions between at least three of the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites. Mutations in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmuscle cells. Therefore, these elements function as muscle-specific splicing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from the alternative exon is conserved in the rat and chicken cTNT genes. A related sequence is found in a third muscle-specific gene, that encoding skeletal troponin T, downstream from an alternative exon with a developmental pattern of alternative splicing similar to that of rat and chicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number of different genes.     

Autoregulation of Fox protein expression to produce dominant negative splicing factors

The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxΔRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxΔRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform.     

Smooth muscle alternative splicing induced in fibroblasts by heterologous expression of a regulatory gene.

Alternative splicing is a common mechanism for regulating gene expression in different cell types. In order to understand this important process, the trans-acting factors that enforce the choice of particular splicing pathways in different environments must be identified. We have used the rat alpha-tropomyosin gene as a model system of tissue-specific alternative splicing. Exon 3 of alpha-tropomyosin is specifically inhibited in smooth muscle cells allowing the alternative inclusion of exon 2. We have used a novel gene transfer and selection strategy to detect a gene whose expression in fibroblasts is sufficient to switch them to smooth muscle-specific splicing of alpha-tropomyosin and also alpha-actinin. Extracts from the regulating fibroblasts contain an apparently novel 55 kDa protein which binds to RNA elements required for regulation of tropomyosin splicing. This protein is not detected in extracts of non-regulating cells and is therefore a strong candidate cell-specific splicing regulator. These experiments advance our understanding of smooth muscle splicing regulation as well as establishing a means for direct cloning of tissue-specific splicing regulators which have so far been refractory to biochemical analysis.     

Competition of PTB with TIA proteins for binding to a U-rich cis-element determines tissue-specific splicing of the myosin phosphatase targeting subunit 1

A considerable amount of smooth muscle phenotypic diversity is generated by tissue-specific and developmentally regulated splicing of alternative exons. The control mechanisms are unknown. We are using a myosin phosphatase targeting subunit-1 (MYPT1) alternative exon as a model to investigate this question. In the present study, we show that the RNA binding proteins TIA and PTB function as antagonistic enhancers and suppressors of splicing of the alternative exon, respectively. Each functions through a single U-rich element, containing two UCUU motifs, just downstream of the alternative exon 5' splice site. Tissue-specific down-regulation of TIA protein in the perinatal period allows PTB to bind to the U-rich element and suppress splicing of the alternative exon as the visceral smooth muscle acquires the fast-phasic smooth muscle contractile phenotype. This provides a novel role for PTB in the tissue-specific regulation of splicing of alternative exons during the generation of smooth muscle phenotypic diversity.