Role of Nova-1 in regulating α2N, a novel glycine receptor splice variant, in developing spinal cord neurons

Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT-PCR, we investigated the regulation of GlyR α-subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR α2 subunit, α2A and α2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named "α2N." Examination of the RNA from spinal cords of different-aged rats showed a dramatic down-regulation of α2N during prenatal development: α2N mRNA formed a significant portion of the α2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR α2 pre-mRNA, the neurooncological ventral antigen-1 (Nova-1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of α2N, calling into question their involvement in the developmental regulation of α2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova-1 variants significantly altered the relative level of GlyR α2N, showing that Nova-1 isoforms can regulate GlyR α2 pre-mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR α2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova-1 and brPTB in the developing spinal cord, and suggest that Nova-1 plays a role in regulating GlyR α2N in developing neurons.