The AS event generating selleck screening library the Gls-l and Gls-s isoforms was listed as a top target in our Aspire2 AS analysis, with a validated ΔI of −0.3, (Figure 6B and Table S7). Quantitative RT-PCR using primers specific for each Gls isoform demonstrated that in Elavl3−/−;Elavl4−/− DKO brain, abundance of the Gls-s isoform did not change while abundance of the Gls1-l isoform was reduced to approximately 50% of the WT levels ( Figure 6D). Western blot analysis using an antibody recognizing a common epitope to both isoforms also demonstrated that the abundance of Gls-s and Gls-l proteins were reduced to 60% and 25% of the WT levels, respectively ( Figures

6C and 6E). Since Elavl3/4 DKO die at age P0 it is difficult to further carry out any physiological analyses. We assessed whether Elavl3−/− single KO mice also exhibited a defect in glutamate regulation and observed a smaller but significant decrease in

total glutamate levels and in Gls-l, but not Gls-s, protein levels ( Figure S5). These results point to a role for nElavl proteins in directly controlling Gls-s and Panobinostat purchase Gls-l levels in the nervous system through reinforcing mechanisms of involving both the regulation of AS and mRNA half-life, consistent with nElavl HITS-CLIP results demonstrating direct binding to both intronic and 3′UTR elements. To assess whether there might be a physiologic correlate of excitation/inhibition imbalance manifested by misregulation of glutamate signaling in Elavl3−/− mice, we undertook an EEG analysis of cortical function. Video EEG monitoring of awake and behaving mutants revealed a striking pattern of abnormal cortical hypersynchronization in both Elavl3+/− and Elavl3−/− mice never seen in WT mice ( Figure 7A; through Movie S1). In Elavl3+/− mice, there was a nearly continuous presence (1–9/min) of bilaterally synchronous sharp cortical

spike discharges, sometimes accompanied by brief afterdischarges ( Figure 7B). Elavl3−/− mice displayed similar discharges as well as more severe, non-convulsive electrographic seizures lasting from 10–30 s ( Figure 7C). Both patterns demonstrate aberrant hypersynchronization in cortical networks. Until recently studies aimed at identifying regulatory RNA sequences have been limited to correlative information lacking direct functional links to biological processes. HITS-CLIP technique provides a methodology to identify such functional RNA-protein interaction sites and has been successfully applied to identifying binding sites and uncovering new biological functions for several RNABPs, including Nova (Licatalosi et al., 2008), PTB (Xue et al., 2009), hnRNP C (König et al., 2010), TIA-1 (Wang et al., 2010b), TDP-43, and Fox2 (Yeo et al., 2009).