A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins t hat participate in the control of alternative splicing. We have used the ye ast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-bin ding protein (PTB), The expression of this protein, brPTB, is enriched in t he brain, where it is expressed in glia and neurons. brPTB interacts with N ova proteins in cell lines and colocalizes with Nova within neuronal nuclei . We previously found that Nova binds to a pyrimidine-rich RNA element pres ent upstream of an alternatively spliced exon, E3A, in glycine receptor alp ha 2 (GlyR alpha 2) pre-mRNA, and this binding is implicated in Nova-depend ent regulation of splicing. Cotransfection assays with a GlyR alpha 2 minig ene demonstrate that brPTB antagonizes the action of Nova to increase utili zation of GlyR alpha 2 E3A, brPTB binds to a 90-nt GlyR alpha 2 RNA adjacen t to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyR alpha 2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dep endent exon selection disappears, These results suggest that brPTB is a tis sue-restricted RNA binding protein that interacts with and inhibits the abi lity of Nova to activate exon selection in neurons.