Alternative splicing of fibronectin mRNAs in chondrosarcoma cells: Role of far upstream intron sequences

Abstract

The fibronectin (FN) gene encodes multiple mRNAs through the process of alternative splicing, and production of certain isoforms is characteristic of a given cell type. Chondrocytes produce FNs that completely lack alternative exon EIIIA, and loss of inclusion of the exon is tightly linked to chondrogenic condensation of mesenchymal cells. The inclusion of a second exon, EIIIB, is high in embryonic cartilage, but declines with age. Multiple exons are omitted to produce the (V + C)-form that is highly specific for cartilage and chondrocytes. A rat chondrosarcoma cell line, RCS, was identified that preserves key features of the cartilage-specific splicing phenotype. RCS cells, which exclude exon EIIIA, and HeLa cells, which include exon EIIIA similar to mesenchymal cells, were used to assess the contribution of intron sequences flanking exon EIIIA to splicing regulation. Deletion of most of the intron downstream of the exon had little effect on splicing in either cell type. However, deletions within upstream intron 32-A reduced inclusion of the alternative exon in both cell types. The sequences involved lie more than 200 nucleotides away from the exon, but could not be localized to a single region by deletion mapping. These intronic sequences contribute to the efficiency of exon EIIIA recognition, but not to cell-type specific regulation. The normally inhibitory factor polypyrimidine tract binding protein promotes exon EIIIA inclusion in a manner that is partially dependent on the regulatory sequences within intron 32-A.

Exon repression by polypyrimidine tract binding protein

“…In systems of alternative splicing, PTB often serves as a repressor of highly tissuespecific exons outside of a particular cell type, such as muscle cells or neurons (Wagner and Garcia-Blanco 2001;Black and Grabowski 2003). Many mammalian alternative exons are known to be affected by the action of PTB, including exons in actinin (Southby et al 1999;Gromak et al 2003), tropomyosin (Mulligan et al 1992;Gooding et al 1994Gooding et al , 1998Pérez et al 1997a), troponin (Charlet et al 2002), c-src (Chan and Black 1997;Chou et al 2000), fibronectin (Norton 1994;Flanagan et al 2003), FGF receptors 1 and 2 (Carstens et al 2000;Jin et al 2003), and IgM (Shen et al 2004). In all of these transcripts, PTB binds to short pyrimidine-rich elements within the RNA encompassing the regulated exon.…”

A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10

“…Our preliminary data also showed that at least two additional minor distal cis elements in intron 9 contributing, respectively, to exon 10 exclusion may exist (data not shown). Although distal intronic cis elements to regulate alternative splicing have been described (Modafferi and Black 1997;Flanagan et al 2003;Li et al 2003), they are usually weak and more difficult to be identified because they are located far from exons and are less accessible to spliceosome and other machineries required for splicing (Graveley et al 1998). We were unable to pinpoint the exact sequences in introns 9 and 10 in this report.…”

Effect of Cryoablation Treatment on the Protein Expression Profile of Low-Grade Central Chondrosarcoma Identified by LC-ESI-MS/MS

“…81 In addition, it also plays a significant role in tumor cell microenvironment. 77 The identified contrast expressions among the two studies illustrate a novel detail that, although both OGS and CSA are primary bone tumors that grow at different life stages and mature in various timelines, they regulate differentially in the tumors of both untreated groups. The higher expressions of FN in the OGS-untreated group indicate tumor cell proliferation and differentiation, etc., in the growing bones of children, which should be suppressed by therapy.…”

“…Mounting evidence reported that FN is a crucial ECM protein widely distributed and mediates numerous cellular interactions such as cell adhesion, migration, cartilage growth regulation, and differentiation. , It regulates critical mechanisms by interacting with ECM molecules such as collagens, integrins, fibrins, etc., Moreover, FN from young, growing, and adult cartilage suggests regulating the cartilage differentiation in tumors . In addition, it also plays a significant role in tumor cell microenvironment …”