Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing

Alternative splicing of fibroblast growth factor receptor-2 (FGFR2) mutually exclusive exons IIIb and IIIc results in highly cell-type-specific expression of functionally distinct receptors, FGFR2-IIIb and FGFR2-IIIc. We previously identified an RNA cis-element, ISE/ISS-3, that enhanced exon IIIb splicing and silenced exon IIIc splicing. Here, we have performed comprehensive mutational analysis to define critical sequence motifs within this element that independently either enhance splicing of upstream exons or repress splicing of downstream exons. Such analysis included use of a novel fluorescence-based splicing reporter assay that allowed quantitative determination of relative functional activity of ISE/ISS-3 mutants using flow cytometric analysis of live cells. We determined that specific sequences within this element that mediate splicing enhancement also mediate splicing repression, depending on their position relative to a regulated exon. Thus, factors that bind the element are likely to be coordinately involved in mediating both aspects of splicing regulation. Exon IIIc silencing is dependent upon a suboptimal branchpoint sequence containing a guanine branchpoint nucleotide. Previous studies of exon IIIc splicing in HeLa nuclear extracts demonstrated that this guanine branchsite primarily impaired the second step of splicing suggesting that ISE/ISS-3 may block exon IIIc inclusion at this step. However, results presented here that include use of newly developed in vitro splicing assays of FGFR2 using extracts from a cell line expressing FGFR2-IIIb strongly suggest that cell-type-specific silencing of exon IIIc occurs at or prior to the first step of splicing.     

Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons

Splicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function. Thus hnRNP M, along with other members of the hnRNP family of RNA-binding proteins, plays a combinatorial role in regulation of FGFR2 alternative splicing. We also determined that hnRNP M can affect the splicing of several other alternatively spliced exons. This activity of hnRNP M included the ability not only to induce exon skipping but also to promote exon inclusion. This is the first report demonstrating a role for this abundant hnRNP family member in alternative splicing in mammals and suggests that this protein may broadly contribute to the fidelity of splice site recognition and alternative splicing regulation.     

Identification of an intronic splicing enhancer essential for the inclusion of FGFR2 exon IIIc

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human beta-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5'-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements.     

Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb

The cell type-specific alternative splicing of FGFR2 pre-mRNA results in the mutually exclusive use of exons IIIb and IIIc, which leads to critically important differences in receptor function. The choice of exon IIIc in mesenchymal cells involves activation of this exon and repression of exon IIIb. This repression is mediated by the function of upstream and downstream intronic splicing silencers (UISS and DISS). Here we present a detailed characterization of the determinants of silencing function within UISS and DISS. We used a systematic mutational analysis, introducing deletions and substitutions to define discrete elements within these two silencers of exon IIIb. We show that UISS requires polypyrimidine tract-binding protein (PTB)-binding sites, which define the UISS1 sub-element, and an eight nucleotide sequence 5'-GCAGCACC-3' (UISS2) that is also required. Even though UISS2 does not bind PTB, the full UISS can be replaced with a synthetic silencer designed to provide optimal PTB binding. DISS is composed of a 5'-conserved sub-element (5'-CE) and two regions that contain multiple PTB sites and are functionally redundant (DISS1 and DISS2). DISS1 and DISS2 are separated by the activator sequence IAS2, and together these opposing elements form the intronic control element. Deletion of DISS in the FGFR2 exon IIIb context resulted in the near full inclusion of exon IIIb, and insertion of this silencer downstream of a heterologous exon with a weak 5' splice site was capable of repressing exon inclusion. Extensive deletion analysis demonstrated that the majority of silencing activity could be mapped to the conserved octamer CUCGGUGC within the 5'CE. Replacement of 5'CE and DISS1 with PTB-binding elements failed to restore repression of exon IIIb. We tested the importance of the relative position of the silencers and of the subelements within each silencer. Whereas UISS1, UISS2, DISS1, and DISS2 appear somewhat malleable, the 5'CE is rigid in terms of relative position and redundancy. Our data defined elements of function within the ISSs flanking exon IIIb and suggested that silencing of this exon is mediated by multiple trans-acting factors.     

A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.     

5'- and 3'-terminal nucleotides in the FGFR2 ISAR splicing element core have overlapping roles in exon IIIb activation and exon IIIc repression

The cell type-specific, mutually-exclusive alternative splicing of the fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is tightly regulated. A sequence termed ISAR (intronic splicing activator and repressor) has been implicated as an important cis regulatory element in both activation of exon IIIb and repression of exon IIIc splicing in epithelial cells. In order to better understand how this single sequence could have dual roles, we transfected minigenes containing a series of 2-bp mutations in the 18 3′-most nucleotides of ISAR that we refer to as the ISAR core. Transfection of cells with dual-exon (IIIb and IIIc) minigenes revealed that mutation of terminal sequences of the core led to decreased exon IIIb inclusion and increased exon IIIc inclusion. Transfection of cells with single-exon IIIb minigenes and single-exon IIIc minigenes revealed that mutation of terminal sequences of the ISAR core led to decreased exon IIIb inclusion and increased exon IIIc inclusion, respectively. Nucleotides of the ISAR core responsible for exon IIIb activation appear to overlap very closely with those required for exon IIIc repression. We describe a model in which ISAR and a 5′ intronic sequence known as IAS2 form a stem structure required for simultaneous exon IIIb activation and exon IIIc repression.     

A novel form of FGF receptor-3 using an alternative exon in the immunoglobulin domain III

Four distinct FGF receptors were cloned and characterized and it was demonstrated that the ligand binding site of FGF receptors is confined to the extracellular immunoglobulin-like (Ig)-domain 2 and 3. The Ig-domain 3 is encoded by two separate exons: exon IIIa encodes the N-terminal half, and the C-terminal half is encoded by either exon IIIb or IIIc in FGFR1 and FGFR2, whereas FGFR4 is devoid of exon IIIb. Alternative usage of exons IIIb and IIIc determine the ligand binding specificity of the receptor. To analyze the arrangement of these exons in FGFR3 we cloned the genomic sequence between exon IIIa and IIIc of FGFR3 and identified an alternative exon, corresponding to exon IIIb of the FGFR1 and FGFR2. The sequence of this exon shows Ig-domain hallmarks, 44% identity with exon IIIb of other FGF receptors and 36% identity with exon IIIc of FGFR3. Using this exon as a probe for mouse RNA as well as PCR analysis, demonstrated that exon IIIb encodes an authentic form of FGFR3 that is expressed in mouse embryo, mouse skin and mouse epidermal keratinocytes. The results demonstrate that the presence of alternative exons for Ig-domain 3 is a general phenomena in FGFR1, 2 and 3, and represents a novel genetic mechanism for the generation of receptor diversity.     

The nonsense-mediated decay pathway and mutually exclusive expression of alternatively spliced FGFR2IIIb and -IIIc mRNAs

Exons IIIb and IIIc of the FGFR2 gene are alternatively spliced in a mutually exclusive manner in different cell types. A switch from expression of FGFR2IIIb to FGFR2IIIc accompanies the transition of nonmalignant rat prostate tumor epithelial cells (DTE) to cells comprising malignant AT3 tumors. Here we used transfection of minigenes with and without alterations in reading frame and with and without introns to examine how translation affects observed FGFR2 splice products. We observed that nonsense mutations in other than the last exon led to a dramatic reduction in mRNA that is abrogated by removal of downstream introns in both DTE and AT3 cells. The mRNA, devoid of both IIIb and IIIc exons (C1-C2), is a major splice product from minigenes lacking an intron downstream of the second common exon C2. From these observations, we suggest that repression of exon IIIc and activation of exon IIIb inclusion in DTE cells lead to the generation of both C1-IIIb-C2 and C1-C2 products. However, the C1-C2 product from the native gene is degraded due to a frameshift and a premature termination codon caused by splicing C1 and C2 together. Derepression of exon IIIc and repression of exon IIIb lead to the generation of both C1-IIIc-C2 and C1-C2 products in AT3 cells, but the C1-C2 product is degraded. The C1-IIIb-IIIc-C2 mRNA containing a premature termination codon in exon IIIc was present, but at apparently trace levels in both cell types. The nonsense-mediated mRNA decay pathway and cell type-dependent rates of inclusion of exons IIIb and IIIc result in the mutually exclusive expression of FGFR2IIIb and IIIc.     

An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.

The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.     

An Intronic Sequence Element Mediates Both Activation and Repression of Rat Fibroblast Growth Factor Receptor 2 Pre-mRNA Splicing

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) is an example of highly regulated alternative splicing in which exons IIIb and IIIc are utilized in a mutually exclusive manner in different cell types. The importance of this splicing choice is highlighted by studies which indicate that deregulation of the FGF-R2 splicing is associated with progression of prostate cancer. Loss of expression of a IIIb exon-containing isoform of FGF-R2 [FGF-R2 (IIIb)] accompanies the transition of a well-differentiated, androgen-dependent rat prostate cancer cell line, DT3, to the more aggressive, androgen-independent AT3 cell line. We have used transfection of rat FGF-R2 minigenes into DT3 and AT3 cancer cell lines to study the mechanisms that control alternative splicing of rat FGF-R2. Our results support a model in which an important cis-acting element located in the intron between these alternative exons mediates activation of splicing using the upstream IIIb exon and repression of the downstream IIIc exon in DT3 cells. This element consists of 57 nucleotides (nt) beginning 917 nt downstream of the IIIb exon. Analysis of mutants further demonstrates that an 18-nt “core sequence” within this element is most crucial for its function. Based on our observations, we have termed this sequence element ISAR (for intronic splicing activator and repressor), and we suggest that factors which bind this sequence are required for maintenance of expression of the FGF-R2 (IIIb) isoform.     

Imaging the alternative silencing of FGFR2 exon IIIb in vivo

Alternative splicing multiplies genomic coding capacity and regulates proteomic composition. A well-studied example of this plasticity leads to the synthesis of functionally distinct isoforms of the Fibroblast Growth Factor Receptor-2 (FGFR2). The regulation of this isoform diversity necessitates the silencing of FGFR2 exon IIIb, which is mediated by flanking intronic splicing silencers and the polypyrimidine tract binding protein (PTB). To visualize this splicing decision in vivo, we developed mice harboring a green fluorescent protein construct that reports on the silencing of exon IIIb. The animals also harbor a red fluorescent protein reporter of constitutive splicing as an allelic control. This dual reporter system revealed that in various organs and cell types the silencing of exon IIIb required the intronic silencers. In neurons, which do not express PTB, we observed robust silencer-dependent repression of exon IIIb, suggesting that the neural paralog, brain PTB, can take over this function. In the epidermis, however, the intronic silencers were not required for efficient silencing. This work provides a first glimpse at splicing regulation among different cell types in vivo and promises the drafting of an anatomic map of splicing decisions.     

hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc

The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.     

Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing

Alternative splicing in the extracellular domain is a characteristic feature of members of the fibroblast growth factor receptor (FGFR) family. This splicing event generates receptor variants, which differ in their ligand binding specificities. A poorly characterized splice variant is FGFR1-IIIb, recently found to be a functional FGF receptor predominantly expressed in the skin. Here we show that FGFR1-IIIb is expressed in normal and wounded mouse skin. Reduced expression of this type of receptor was found in wounds of healing-impaired genetically diabetic mice, suggesting that downregulation of FGFR1-IIIb is associated with wound healing defects. To address this possibility, we deleted the IIIb exon of FGFR1 in mice. The lack of FGFR-IIIb did not alter the expression of either FGFR1-IIIc, other FGF receptor genes or of FGFR1-IIIb ligands in normal and wounded skin. Histological analysis of the skin of FGFR1-IIIb knockout animals did not reveal any obvious abnormalities. Furthermore, full-thickness excisional skin wounds in these mice healed normally and no defects could be observed at the macroscopic or histological level. Finally, several genes that encode key players in wound repair were normally expressed in these animals. These data demonstrate that FGFR1-IIIb is dispensable for skin development and wound repair.