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

The regulation of eukaryotic gene expression involves an ordered sequence of events within various subcellular compartments. RNA binding proteins (RBPs) play a critical role in this process, acting at multiple levels to regulate and diversify gene expression, and such functions are likely to be of particular importance to neurons. RBPs regulate processes such as alternative splicing, which is an important mechanism for generating complexity in specialized cells, particularly in neurons. For example, all of the major neurotransmitter receptors contain subunits that are alternatively spliced, and these variants can alter receptor localization, ligand binding, signal transduction, and electrophysiological properties (13, 20, 44, 51). Moreover, RBPs and aberrant splicing of mRNAs encoding proteins critical for proper functioning of neurons have been associated with a number of neurological diseases (reviewed in reference 19). To understand the role RBPs play in regulating gene expression in neurons, it would be necessary to identify their complement of RNA targets and to understand how they are recognized amidst the complexity of the cellular environment. Regulation of alternative splicing occurs through recognition of cis-acting sequences in pre-mRNA by trans-acting factors. cis-acting repressor elements that mediate the exclusion of the neuron-specific exons in nonneuronal tissues have been identified through studies of neuron-specific splicing in transcripts such as c-src, the GABAA receptor γ2 (GABAARγ2) subunit, the N-methyl-d-aspartate receptor NR1 subunit, and clathrin light-chain B mRNAs (3, 9, 10, 60, 63, 64). These repressor sequences are mostly pyrimidine rich and, when present in trans as RNA competitors in vitro, stimulate neuronal splicing patterns (10, 64). A number of generally expressed trans-acting factors, including hnRNP H, hnRNP F, KSRP, and PTB (3, 10, 12, 46), have been found to bind such sequences and are believed to play important roles in regulating alternative splicing. In addition to the contribution of generally expressed factors, it has long been suspected that tissue-specific factors regulate alternative splicing in the brain (1). Study of the paraneoplastic neurologic disorders (PNDs) has resulted in the identification of Nova-1 (7), a neuron-specific protein involved in splicing regulation (32). PNDs are rare immune-mediated disorders associated with ectopic expression of neuron-specific proteins in tumors (reviewed in references 15 and 48). Patients harbor characteristic high titers of antibodies specific for these antigens in their sera, and immune targeting of these antigens in the central nervous system (CNS) is thought to result in the neuronal death and neurological symptoms seen in PNDs. Nova proteins are the target antigens in paraneoplastic opsoclonus-myoclonus ataxia, a PND characterized by loss of inhibitory control of motor neurons in the spinal cord and brainstem. The Nova proteins are RBPs containing 3 KH-type (for hnRNP K homology) RNA binding domains. Widely expressed KH domain-containing proteins have been shown to play roles in a number of aspects of RNA processing, including mRNA stability and translational control (hnRNP K and hnRNP E1/E2) (35, 49), subcellular localization of RNAs to specific sites in the cell cytoplasm (ZBP-1 and Vera) (17, 55), and RNA splicing (KSRP [46] and SF1 [2] in mammals, PSI [57] in Drosophila, and MER-1 [21] in Saccharomyces cerevisiae). Nova-1 is expressed exclusively in neurons of the CNS, including the brainstem and spinal motor neurons targeted in paraneoplastic opsoclonus-myoclonus ataxia. Nova-1 null mice display a severe phenotype. Although they appear normal at birth, they are markedly smaller than their wild-type littermates at as little as 3 to 4 days postbirth and display symptoms of motor impairment such as motor weakness and an action-induced tremor (32). Nova proteins act as sequence-specific RNA binding proteins. A consensus binding sequence determined by in vitro RNA selection consists of a stem-loop RNA containing (UCAUY)3 in the loop region (6). This binding is sequence specific, as a number of discrete nucleotide substitutions, such as mutagenesis of this core to (UCUUY)3, abolish RNA binding. Studies using the third Nova KH domain in isolation identified high-affinity targets containing a single UCAY element, again within a hairpin loop context (33), and allowed solution of the X-ray structure of a Nova KH3/RNA cocrystal. The crystal structure reveals that the RNA stem-loop presents the UCAY sequence to a “molecular vise” formed by a cleft between the variable and invariant protein loops of the Nova KH3 domain (42). In particular, the CA dinucleotide forms contacts with the protein to create pseudo-Watson-Crick base pairs and predicts that these residues are absolutely necessary for binding to occur. The structure agrees precisely with Nova KH3 domain-RNA binding studies in which RNA mutations still conforming to the consensus HYCAY (where H denotes A, C, or U and Y denotes C or U) are tolerated (33). These studies led to the identification of GlyRα2 inhibitory neurotransmitter receptor subunit pre-mRNA as a Nova target RNA. Nova binds this transcript 80 nucleotides (nt) upstream of an alternatively spliced exon, E3A, which is spliced in a mutually exclusive manner with a downstream exon, E3B. These exons encode part of the extracellular domain of the receptor and thus may be involved in ligand binding (39). Nova protein in mouse brain lysates coimmunoprecipitates with GlyRα2 pre-mRNA (6) and can be UV cross-linked to an RNA encoding a region of the GlyRα2 intronic sequence (32). Cotransfection of GlyRα2 minigene constructs with a Nova-1 expression plasmid demonstrated that Nova-1 is able to act directly on GlyRα2 pre-mRNA to enhance the inclusion of GlyRα2 E3A and that this action is dependent on the integrity of the intronic (UCAU)3 Nova binding site (32). Analysis of GlyRα2 alternative splicing in vivo provided genetic support for these conclusions, as Nova-1 null mice showed a decrease in utilization of GlyRα2 E3A relative to wild-type littermates (32). This defect in alternative splicing regulation was restricted to regions that normally express Nova-1 and was specific. Regulation of a number of alternatively spliced RNAs was unaffected in Nova-1 null mice, with one notable exception: alternative splicing of exon 9 in the inhibitory neurotransmitter receptor subunit pre-mRNA, GABAARγ2, was disrupted in Nova-1 null mice in a dose-dependent manner, and these defects were again restricted to regions which normally express Nova-1. Alternative splicing of GABAARγ2 cassette exon E9 generates two mRNA isoforms, γ2L (long [E9 included]) and γ2S (short), which show differential spatial and temporal expression characteristics (24, 27, 59). Inclusion of E9 adds 8 amino acids to an intracellular loop of the protein which contain a consensus site for phosphorylation by protein kinase C (37, 61). Phosphorylation of this site has been shown to modulate GABAAR function (38). Mice engineered to express only the short form of the GABAARγ2 subunit display a higher level of anxiety than control mice (30) and increased sensitivity to benzodiazepines (53). Studies aimed at trying to understand the mechanism behind regulation of GABAARγ2 splicing have shown that nucleotides within the exon and the adjacent splice sites are essential for regulation in a neuronal versus a nonneuronal cell line (63). PTB has been shown to inhibit γ2L splicing, leading to the hypothesis that splicing is regulated by a mechanism involving derepression of splicing at this site in neurons of the CNS (3, 64). However, it is unknown whether these observations are relevant to γ2L splicing in neurons and/or whether they involve Nova protein. To understand how Nova acts to regulate GABAARγ2 splicing, we have determined the sequence requirements for Nova-1 regulation of E9 inclusion. We find that previously mapped elements important for the regulation of splicing in tissue culture are not involved in Nova regulation of γ2L. Remarkably, a broad and unbiased screen for regulatory elements over 3.7 kb of GABAARγ2 pre-mRNA revealed that a single intronic YCAY repeat element is necessary and sufficient to mediate Nova-dependent regulation. These studies define Nova as a neuron-specific splicing regulator that functions through action on a newly identified intronic splicing enhancer which lies far downstream of the regulated exon.