Abstracts

BRUNOL4 (Bruno like 4) is a brain specific RNA binding protein, the loss of which leads to both convulsive and absence seizures in mice. The expression of Brunol4 is concentrated in neurons of the brain regions controlling synchronization and oscillation such as cerebral cortex, hippocampus and thalamus, suggesting that BRUNOL4 may play important roles in the maintenance of normal neuronal rhythmic activities. We hypothesize that BRUNOL4 is involved in the processing of mRNAs encoding proteins critical to neuronal excitation. Thus, BRUNOL4 deficiency would lead to altered expression of several molecules which in turn would result in seizures. This study aims to identify differentially expressed genes in the Brunol4 mutant brains and to determine how BRUNOL4 regulates gene expression., RNAs were extracted from mutant and control brains before the onset of overt seizures. The transcriptomes between mutants and controls were compared by microarrays, followed by northern and western analysis to confirm the expression difference. A potential BRUNOL4 recognition site in the target RNA transcripts was identified by multi-species sequence comparison. Direct binding between BRUNOL4 and its RNA targets was demonstrated by RNA-immunoprecipitation in neurons. The consequences of the BRUNOL4-RNA interactions were investigated through reporter assays and RNA decay studies in cultured neurons., Multiple potential BRUNOL4 target RNAs with altered expression were identified in mutants. Subsequent work confirmed the reduced expression of four RNAs encoding proteins critical to neuronal excitation in the mutant brain. The RNAs encode N-ethylmaleimide-sensitive factor, serotonin receptor 2c, synapsin II and [alpha]-synuclein, some of which have been implicated in seizures while others may represent novel pathways in epilepsy. Sequence analysis revealed a highly conserved U/G rich motif in the 3[apos] untranslated region (UTR) of all four genes. RNA- immunoprecipitation showed that BRUNOL4 interacts with the 3[apos] UTRs of the four RNA transcripts through binding to the conserved U/G rich region. In luciferase reporter assays, the expression of chimeric reporters containing 3[apos] UTRs of the target RNAs was down-regulated in the Brunol4 mutant neurons compared with wildtype neurons, a defect that can be rescued by adding back BRUNOL4 through transfection. Preliminary studies suggest that the mRNA decay rates of the four RNA transcripts are shorter in mutant neurons., BRUNOL4 appears to modulate neuronal excitability at the RNA level through fine-tuning the stability of RNA transcripts encoding proteins critical to neuronal excitation. Brunol4 mutant mice represent the first animal model where compromised mRNA processing leads to epilepsy, thus providing a novel disease mechanism for seizure disorders., (Supported by a research award from Citizens United for Research in Epilepsy (YY) and a grant from NINDS (NS31348 to WNF).)