Nova Regulates GABA_A Receptor γ2 Alternative Splicing via a Distal Downstream UCAU-Rich Intronic Splicing Enhancer

Abstract

Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABA_A receptor γ2 (GABA_ARγ2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABA_ARγ2 alternative splicing, we systematically screened minigenes derived from the GABA_ARγ2 and human β-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABA_ARγ2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Nova-dependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABA_ARγ2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.

ACKNOWLEDGMENTS

We thank A. R. Krainer for the sp64-HβΔ6 plasmid from which the β-globin constructs were derived. We are grateful to all members of the laboratory, especially K. B. Jensen and J. C. Darnell, for advice and helpful discussions and for critical reading of the manuscript.

B.K.D. was supported in part by a B. Duke Glenn Fellowship. This research was supported by the Howard Hughes Medical Institute (B.K.D. and R.B.D.), the Burroughs Wellcome Fund, and NIH R01 NS40955 and NS34389 (R.B.D.).