Elements regulating an alternatively spliced exon of the rat fibronectin gene.

We have investigated the regulation of splicing of one of the alternatively spliced exons in the rat fibronectin gene, the EIIIB exon. This 273-nucleotide exon is excluded by some cells and included to various degrees by others. We find that EIIIB is intrinsically poorly spliced and that both its exon sequences and its splice sites contribute to its poor recognition. Therefore, cells which recognize the EIIIB exon must have mechanisms for improving its splicing. Furthermore, in order for EIIB to be regulated, a balance must exist between the EIIIB splice sites and those of its flanking exons. Although the intron upstream of EIIIB does not appear to play a role in the recognition of EIIIB for splicing, the intron downstream contains sequence elements which can promote EIIIB recognition in a cell-type-specific fashion. These elements are located an unusually long distance from the exon that they regulate, more than 518 nucleotides downstream from EIIIB, and may represent a novel mode of exon regulation.     

Subcellular and developmental expression of alternatively spliced forms of fibroblast growth factor 14

Fibroblast growth factors (FGFs) 11–14 comprise a subfamily of FGFs with poorly defined biological function. Here we characterize two isoforms of FGF14 (FGF14-1a and FGF14-1b) that result from the alternative usage of two different first exons. We demonstrate that these isoforms have differential subcellular localization and that they are differentially expressed in various adult tissues. Using in situ hybridization we show that Fgf14 is widely expressed in brain, spinal cord, major arteries and thymus between 12.5 and 14.5 days of mouse embryonic development. We also show that during cerebellar development, Fgf14 is first observed at postnatal day 1 in post mitotic granule cells, and later in development, in migrating and post migratory granule cells. The developmental expression pattern of Fgf14 in the cerebellum is complementary to that of Math1, a marker for proliferating granule cells in the external germinal layer.     

Spatial expression of the alternatively spliced EIIIB and EIIIA segments of fibronectin in the early chicken embryo

Using domain-specific antibodies, we have analyzed the tissue distribution of fibronectins (FNs) containing the alternatively spliced EIIIB and EIIIA segments relative to total FN in early chicken embryos. The results show a selective loss of EIIIA+ FN staining in the notochordal sheath and in cartilaginous structures between 4.5 and 7.0 days of development. In other regions, EIIIB+ and EIIIA+ FNs are extensively codistributed in and around mesoderm-derived structures (somites, notochord, heart, and blood vessels), in basal laminae of endoderm and ectoderm-derived structures, as well as within the vicinity of neural crest formation and migration. We also noted that EIIIA staining overlaps with spatial patterns of distribution that have previously been described for the alpha4 integrin subunit, a component of the EIIIA receptor alpha4beta1.     

Multiple cardiovascular defects caused by the absence of alternatively spliced segments of fibronectin

Alternatively spliced variants of fibronectin (FN) containing exons EIIIA and EIIIB are expressed around newly forming vessels in development and disease but are downregulated in mature vasculature. The sequences and patterns of expression of these splice variants are highly conserved among vertebrates, suggestive of their biological importance; however the functions of EIIIA and EIIIB-containing FNs are unknown. To understand the role(s) of these splice variants, we deleted both EIIIA and EIIIB exons from the FN gene and observed embryonic lethality with incomplete penetrance by embryonic day 10.5. Deletion of both EIIIA and EIIIB exons did not affect synthesis or cell surface deposition of FN, indicating that embryonic lethality was due specifically to the absence of EIIIA and EIIIB exons from FN. EIIIA/EIIIB double-null embryos displayed multiple embryonic cardiovascular defects, including vascular hemorrhage, failure of remodeling embryonic and yolk sac vasculature, defective placental angiogenesis and heart defects. In addition, we observed defects in coverage and association with dorsal aortae of alpha-smooth-muscle-actin-positive cells. Our studies indicate that the presence or absence of EIIIA and EIIIB exons alters the function of FN and demonstrate the requirement for these alternatively spliced exons in cardiovascular development.     

The Drosophila Ret gene is transcribed in multiple alternatively spliced forms

We have cloned and sequenced the 5' and 3' ends of the Drosophila homolog of the vertebrate c-ret gene, Ret, and have derived from it the predicted protein sequence of Ret. The extracellular domain of Ret is very widely diverged from that of its vertebrate counterparts but the cadherin motif present in vertebrate c-ret proteins can also be discerned in Ret. As with the vertebrate gene, multiple splice variants were detected at the 5'-end of Ret, one of which inserts an exon with a protein-terminating frameshift into the cDNA. In contrast to human c-ret, which may vary its signalling specificity by using splicing-derived, alternative C-terminal sequences, Ret cDNAs showed no variation at their 3'-ends.     

The alternatively spliced type III connecting segment of fibronectin is a zinc-binding module.

Fibronectin (FN) is a prototypic adhesive glycoprotein that is widely expressed in extracellular matrices and body fluids. The fibronectin molecule is dimeric, and composed of a series of repeating polypeptide modules. A recombinant fragment of FN incorporating type III repeats 12-15, and including the alternatively-spliced type three connecting segment (IIICS), was found to bind Ni(2+), Cu(2+) and Zn(2+) divalent cations, whereas a similar fragment lacking the IIICS did not. Mutation of two pairs of histidine residues in separate spliced regions of the IIICS reduced cation binding to near the level of the variant lacking the IIICS, suggesting a zinc finger-like mode of cation coordination. Analysis of native FNs purified from plasma or amniotic fluid revealed significant levels of zinc associated with those isoforms that contain the complete IIICS. Taken together, these data demonstrate that the IIICS region of FN is a novel zinc-binding module.     

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man

Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.     

Characterization and expression of a novel alternatively spliced human angiopoietin-2

Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2(443) (Ang2(443)), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96-148) is absent in Ang2(443) because of alternative splicing of the gene. Like Ang2, recombinant Ang2(443) expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2(443) binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2(443) on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression of Ang2(443) mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2(443) mRNA and protein. Macrophages express mainly Ang2 mRNA, but the expression of Ang2(443) mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2(443) is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.     

Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization

Cytotactin, an extracellular glycoprotein found in neural and nonneural tissues, influences a variety of cellular phenomena, particularly cell adhesion and cell migration. Northern and Western blot analysis and in situ hybridization were used to determine localization of alternatively spliced forms of cytotactin in neural and nonneural tissues using a probe (CT) that detected all forms of cytotactin mRNA, and one (VbVc) that detected two of the differentially spliced repeats homologous to the type III repeats of fibronectin. In the brain, the levels of mRNA and protein increased from E8 through E15 and then gradually decreased until they were barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridized only to the 7.2-kb message. In isolated cerebella, the 220-kD polypeptide and 7.2-kb mRNA were the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message that contains the VbVc alternatively spliced insert. In situ hybridization showed cytotactin mRNA in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs were more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA was observed in the soma whereas the protein was present externally along the glial fibers. In the telencephalon, cytotactin mRNAs were found in a narrow band at the edge of a larger region in which the protein was wide-spread. Hybridization with the VbVc probe generally overlapped that of the CT probe in the spinal cord and cerebellum, consistent with the results of Northern blot analysis. In contrast, in the outermost tectal layers, differential hybridization was observed with the two probes. In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, was detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes was observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme expressed cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells expressed cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contained cytotactin mRNA. These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissue-specific local signals and prompt a search for functional differences in the various molecular forms of the protein.     

Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts

There are three types of mouse Mutyh mRNAs (type a, b and c) generated by alternative splicing, and type b mRNA is a major form among the three in most of the tissues examined. The level of type c mRNA is relatively high in brain. Type a and b mRNAs were expected to encode 57.7 kDa protein (MUTYHalpha), while type c mRNA had a partly different open reading frame encoding a 50.2 kDa protein (MUTYHbeta). An in vitro translation of type b and c mRNAs produced a 50 kDa MUTYHalpha and 47 kDa MUTYHbeta, respectively. MUTYHalpha and MUTYHbeta were detected in wild-type embryonic stem (ES) cells or thymocytes prepared from wild-type mice, but neither MUTYH-null ES cells nor thymocytes prepared from MUTYH-null mice. Both MUTYHalpha and MUTYHbeta were mainly localized in the nuclei and some in mitochondria in wild-type ES cells. Recombinant MUTYHalpha and beta were expressed as fusion proteins with thioredoxin in Escherichia coli, but only MUTYHalpha was partly soluble and thus could be purified. Recombinant MUTYHalpha possessed DNA glycosylase activities to excise adenine opposite 8-oxoguanine and guanine but not AP lyase activity.