Tra2βl regulates P19 neuronal differentiation and the splicing of FGF‐2R and GluR‐B minigenes

Abstract

The present study demonstrates that the expression of Tra2beta1 (Transformer 2-beta1) proteins, an SR (serine/arginine rich) protein, is developmentally up-regulated in a neural-specific pattern. The up-regulation is also observed in RA (retinoic acid) induced neural differentiation of P19 cells. Tra2betal proteins are located in the nuclei of P19 cells, which are consistent with its functional site as an SR protein. The over-expression of Tra2betal proteins promotes RA induced neuronal differentiation of P19 cells. In P19 cells, the splicing of FGF-2R (fibroblast growth factor receptor 2) minigene produces the BEK form, while the alternative splicing of GluR-B (glutamate receptor subunit B) minigene generates two products, the Flop and the Truncated isoforms. Tra2betal inhibits the BEK splicing, but it promotes the Flop splicing. The results therefore suggest that Tra2betal is involved in the regulation of alternative splicing processes during neural development, peculiarly the splicing of FGF-2R and GluR-B genes. Both FGF-2R and GluR-B genes are known to play important roles in neural differentiation.

Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation

“…P19 is a teratocarcinoma-derived pluripotent stem cell line that can give rise to all three germ layers in mice (23). Undifferentiated P19 EC cells in culture can be induced by RA to differentiate to neuronal and glial cells (24,25) or by DMSO to cardiac and skeletal muscles (26). Treatment of P19 EC cells with RA for up to 4 days induces non-adhering aggregates called EBs which resemble the inner cell mass of embryos (27,28).…”

Expression Pattern and Splicing Function of Mouse ZNF265

“…The transcript variants spliced from the minigenes are expected to be Flop (612 bp) and Flip (612 bp), respectively, in neurons. But, in cultured cells, the minigenes are expected to generate Flop and truncated (500 bp) transcript isoforms [8]. The truncated GluR-B transcript contains neither Flip nor Flop exon, as illustrated in Fig.…”

“…The Flop and truncated isoforms from the GluR-B minigene, as indicated in Fig. 4A, were identified using enzymatic digestion of the transcripts as described previously (8). The in vivo splicing analysis showed that overexpression of ZNF265-1 significantly decreased the ratio of Flop to truncated isoforms (Fig.…”

“…The primers for GluR-B minigene products were described previously [8]. For detection of SMN2 minigene products, to avoid the interference from the endogenous genes, a primer specific to the vector DNA sequence (T7 primer) and a primer specific to SMN (5 0 -GCT TCA CAT TCC AGA TC-3 0 ) were used.…”

“…The SMN2 (survival motor neuron 2) minigene containing a SMN genomic DNA fragment from exon 6 to exon 8 in the mammalian expression vector pCI (a gift from professor Brunhilde Wirth, Institute of human Genetics, University of Bonn, Germany) and GluR-B (glutamate receptor subunit B) minigene containing a GluR-B genomic DNA fragment [exon 13 and two alternatively spliced exons, exon14 (Flop exon), exon15 (Flip exon) and exon 16] were used for in vivo splicing assays as described previously [7][8][9][10]. In brief, for each 3 cm culture dish, 2 lg minigene vectors and 0 or 2 lg of ZNF265-1-His-tag expression plasmids were co-transfected into COS1 cells using lipofectamine 2000.…”