Nova regulates GABA(A) receptor gamma2 alternative splicing via a distal downstream UCAU-rich intronic splicing enhancer.
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 gamma2 (GABA(A)Rgamma2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABA(A)Rgamma2 alternative splicing, we systematically screened minigenes derived from the GABA(A)Rgamma2 and human beta-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABA(A)Rgamma2 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(A)Rgamma2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.
Dredge
B Kate
BK
Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.
Darnell
Robert B
RB
D017398:Alternative Splicing; D000818:Animals; D000951:Antigens, Neoplasm; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003101:Collodion / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004742:Enhancer Elements, Genetic; D005091:Exons; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics; D006801:Humans; D007438:Introns; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D000090682:Neuro-Oncological Ventral Antigen; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D000090682:Neuro-Oncological Ventral Antigen; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D005914:Globins; D003101:Collodion