Fig 2: T-STAR mediates splicing repression of Nrxn2 AS4 via a downstream response element.(A) Nucleotide sequence immediately downstream of the Nrxn2 alternative exon (exon sequence shaded grey, intron sequence unshaded) of wild type gene and after mutagenesis to remove U((U/A)AA motifs. (B) Capillary gel electrophoretogram to show splicing response of mutated Nrxn2 minigene after co-transfection with the indicated expression constructs. (C) Percentage Splicing Exclusion levels of the mutated Nrxn2 minigene from 3 independent transfection experiments. The p values were calculated using unpaired t tests, and estimate statistical significance between splicing exclusion of the mutated Nrxn2 minigene on co-expression of GFP(lane 1: shown as green bar) and each of the individual fusion proteins (lanes 2,4–7: shown as black bars). (D) Coomassie stained gel showing purified Sam68-GST and T-STAR-GST fusion proteins used for gelshift experiments. (E) EMSA experiment using RNA probe containing the Nrxn2 response element. (F) EMSA experiment using a control RNA probe.

Fig 3: Nrxn2 is a specific splicing target for T-STAR but not Sam68.(A) Comparative modular organisations of the Sam68 and T-STAR proteins. The position of the serine residue 20 (S20) in Sam68 which is phosphorylated to mediate splicing regulation of Nrxn1 AS4 is indicated. (B) Representative Western blot analysis of HEK293 cells co-transfected with expression constructs for GFP-fusion proteins and minigenes. The Western blot was probed for GFP and actin, showing equal expression levels of each fusion protein. (C) Nrxn2 AS4 is regulated by T-STAR but not Sam68. Representative capillary gel electrophoretogram (top) and bar chart (bottom) showing splicing data from single and three independent transfections respectively. (D) Nrxn3 splicing is regulated by T-STAR and Sam68. Representative capillary gel electrophoretogram (top) and bar chart (bottom) showing splicing data from single and three independent transfections respectively. In each case, statistical significance was compared between HEK293 cells expressing GFP (lane1; shown as green bar) and HEK293 cells expressing the GFP fusion proteins (lanes 2–8 shown as black bars) using a non-paired t test and the error bar represents the standard error of the mean.

Fig 4: T-STAR protein concentration correlates with Nrxn1-3 AS4 alternative splicing patterns.(A) Expression levels of T-STAR and Sam68 protein in different regions of the mouse brain were measured using Western blotting. The same filters were first probed with antisera specific for T-STAR, and then stripped and reprobed with an antisera specific for Sam68. (B–D) Levels of Nrxn1-3 AS4 Percentage Splicing Exclusion in each brain region plotted against the ratio of T-STAR: Sam68 protein quantified from the Western blot shown in (A). The dashed line is the 95% confidence limit of the best fit line.

Fig 5: T-STAR protein regulates region-specific splicing of Neurexin1-3 AS4 in the mouse brain.(A) Neurexin splicing regulation in different regions of the mouse brain (B) Schematic of the different mouse brain regions used for analysis. (C–E) Percentage splicing exclusion in different regions of the mouse brain (n = 3 mice from each genotype) measured in RNA samples from wild type (column +) and knockout (column -) mice for AS4 of (C) Nrxn1, (D) Nrxn2 and (E) Nrxn3. The error bars correspond to the standard error of the mean. Statistical significances were calculated using a two tailed unpaired t test. No splicing exclusion was observed for Nrxn3 in the absence of T-STAR protein in any brain region.