Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma
Figure 1
(A) Schematic of PAX3, FKHR, PAX3-FKHR and the predicted FKHR-PAX3 protein structures indicating the known functional domains. R: repressor; DBD: DNA binding domain; AD: activation domain. (B) Diagrammatic illustration of the exon-intron organization of human PAX3 gene, and the five alternatively spliced mRNAs that could result from processing of the FKHR-PAX3 primary transcript. PAX3c, PAX3d, and PAX3e use stop codons in intron 8, intron 9, and exon 10. PAX3g and PAX3h are truncated isoforms of PAX3d and PAX3e, respectively, that splice out exon 8. (C) Expression of FKHR-PAX3 transcript isoforms c, d, and e in ERMS (RD) and ARMS (RH4, RH28, RH30) cell lines as detected by RT-PCR and confirmed by Southern hybridization. Top panel: schematic indicates the positions of the FKHR-specific primer (F4) and the isoform-specific PAX3 PCR primer pairs, and the DNA probe spanning the FKHR-PAX3 fusion site used in the Southern analysis are indicated (not to scale). (D) Quantitative RT-PCR analysis of PAX3, FKHR, PAX3-FKHR, and FKHR-PAX3 expression in ARMS cell lines. The relative expression data are presented at two different scales on the Y-axis, high (left panel) and low (right panel) to compensate for the high levels of PAX3-FKHR expression. The relative expression level of PAX3/GAPDH in RH4 cells was assigned an arbitrary value of 1, and used as the reference to calculate fold change. (E) Nucleotide sequences of the cloned FKHR-PAX3 isoforms c, d, and e cDNAs. Top panel: schematic of the FKHR and the isoform-specific PAX3 primer pairs used to clone the full-length protein coding cDNA from RH30 cells. Primer location is approximate for illustrative purposes only. Sequence data is annotated by text in: Plain: 5’ FKHR-UTR; plain/italic: 3’ PAX3-UTR; Bold: protein coding sequences; bold/capital/underline: translation start codon; bold/capital/italic/underline: translational stop codons; underline: isoform-specific sequences.