Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA.
Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell-specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte- and mesenchymal cell-derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type-specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included.
Uporova
T M
TM
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Norton
P A
PA
Tuan
R S
RS
Bennett
V D
VD
D017398:Alternative Splicing; D000818:Animals; D002454:Cell Differentiation; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D020219:Chondrogenesis / Q000235:genetics*; D046508:Culture Techniques; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D005353:Fibronectins / Q000235:genetics*; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014162:Transfection
D017931:DNA Primers; D005353:Fibronectins