Generation of a null <i>Khdrbs3</i> allele.

<p>(A) Northern analysis of different adult mouse tissues to detect expression of <i>Khdrbs3</i> (upper panel) and small subunit rRNA (lower panel). (B) The genomic structure of the <i>Khdrbs3</i> alleles from wild type, floxed, and null mice mice were monitored using Southern blotting and the probe indicated in parts C–D. The Southern blot demonstrates that the cross with a PGK-Cre mouse successfully removed exon 2 from the genomic DNA. (C) Genomic structure of the <i>Khdrbs3^LoxP</i> conditional allele in which exon 2 of the <i>Khdrbs3</i> gene is flanked by <i>Lox</i>P sites. (D) Genomic structure of the null (<i>Khdrbs3⁻</i>) allele from which exon 2 has been deleted by Cre-mediated recombination. (E) Multiplex RT-PCR analysis of <i>Khdrbs3</i> and <i>Hprt</i> mRNA levels in different mouse tissues. The size markers are shown in nucleotides. (F) Western blot analysis of Sam68 and T-STAR protein levels in the testes of wild type and <i>Khdrbs3</i> null mice using an antibody that recognizes T-STAR and Sam68. The position of the size markers are shown in KDa.</p

T-STAR protein regulates region-specific splicing of <i>Neurexin1-3 AS4</i> in the mouse brain.

<p>(A) <i>Neurexin</i> 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) <i>Nrxn1</i>, (D) <i>Nrxn2</i> and (E) <i>Nrxn3</i>. 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 <i>Nrxn3</i> in the absence of T-STAR protein in any brain region.</p

T-STAR protein is a dose-dependent splicing regulator of <i>Nrxn1-3</i> AS4 in the mouse brain.

<p>(A) Agarose gels showing levels of AS4 splicing inclusion from each of the <i>Nrxn1</i>, <i>Nrxn2</i> and <i>Nrxn3</i> genes using three different mice from each genotype. (B) Percentage Splicing Exclusion levels of <i>Nrxn1-3</i> AS4 and <i>Stxbp5l</i> exon 23 measured measured by RT-PCR and capillary gel electrophoresis in wild type (WT), <i>Khdrbs3^+/−</i> (HET) and <i>Khdrbs3^−/−</i> mice (KO) (n = 3 mice for each genotype). The p values were calculated from two tailed unpaired t tests, and error bars represent standard errors of the mean.</p

Concentration-dependent splicing model for regional regulation of <i>Nrxn1-3</i> AS4 in the mouse brain.

<p>T-STAR protein directly regulates <i>Nrxn1-3</i> AS4 splicing. In the cerebellum T-STAR concentrations are low and most of the <i>Nrxn1-3</i> mRNA isoforms include AS4 as a result. In the forebrain-derived regions T-STAR concentrations are high, and there are also increased levels of AS4 splicing exclusion resulting from this. Sam68 protein levels are similar across the brain regions.</p

Human T-STAR protein represses splicing inclusion of the Zebrafish <i>Nrxn3</i> AS4 exon.

<p>(A) Capillary gel electrophoretogram showing splicing of a minigene encoded zebrafish <i>Nrxn3</i> in response to co-expressed proteins introduced by co-transfection. (B) Quantification of biological replicates from three independent co-transfection experiments.</p

T-STAR protein is expressed in the embryonic brain.

<p>(A) Section of embryonic brain (13.5 day embryo) including most of the forebrain region, stained for T-STAR (brown) and counterstained with haematoxylin (blue). (B) <i>Nrxn1-3</i> exon AS4 splicing patterns in wild type and T-STAR knockout 13.5d embryonic brain.</p

T-STAR protein concentration correlates with <i>Nrxn1-3</i> AS4 alternative splicing patterns.

<p>(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 <i>Nrxn1-3</i> 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.</p

<i>Nrxn</i> exon AS4 alternative splicing control is dependent on the physiological expression of T-STAR protein even though Sam68 is co-expressed.

<p>(A) Immunolocalisation of T-STAR and Sam68 proteins in the mouse hippocampus from wild type or knockout mouse brains (Abbreviations: DG - Dentate Gyrus; and AH -Ammon's Horn). The scale bar is equivalent to 20 µm). (B) Immunolocalisation in the mouse testis. Paraffin embedded adult mouse testis sections were stained with affinity purified antibodies raised against T-STAR or Sam68 (brown staining), and counterstained with haematoxylin (blue). Abbreviations: Spg –spermatogonia; Spc –spermatocyte; Rtd –round spermatid; Spd –elongating spermatid; SC –Sertoli cell. The size bar corresponds to 20 µM. (C) Levels of <i>Nrxn1</i> and <i>Nrxn3</i> AS4 alternative splice isoforms in the testes of different mouse genotypes (n = 3 mice of each genotype) measured by RT-PCR and agarose gel electrophoresis. (D) Quantification of Percentage Splicing Exclusion in the testes of different mouse genotypes using capillary gel electrophoresis (n = 3 mice of each genotype: wild type mice <i>Khdrbs3^+/+</i> (abbreviated WT) <i>Khdrbs3^+/−</i> mice (abbreviated HET) and <i>Khdrbs3^−/−</i> mice (abbreviated KO). The p values were calculated using unpaired t tests, to determine the significance of the difference between percentage splicing exclusion levels in the wild type versus either the heterozygous <i>Khdrbs^+/−</i> mice (HET); or wild type versus the homozygous <i>Khdrbs3^−/−</i> (KO) mice. The standard error of the mean is shown as an error bar.</p

Switch-like tissue-specific ASEs are conserved in all vertebrates.

<p>RT-PCR was performed on 15 genes across human, mouse and zebrafish. The 9 genes shown have conserved switch-like splicing in all three vertebrate species. Brain-specific splice forms are indicated with a red arrow. The alternative kidney/liver-specific forms are indicated with a double-headed blue arrow. The different, expected and found, PCR sizes for the long and short form of each gene in each species are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125998#pone.0125998.s005" target="_blank">S1 Table</a>.</p

Nine vertebrate brain specific alternative splice events.

<p><b>a. Primary structures of proteins encoded by the 9 human genes with vertebrate conserved brain-specific splicing.</b> Shown are the annotated human proteins with the regions of the nine splice events indicated by red boxes. TM: transmembrane region; β-APP C-ter: C-terminus of the β Amyloid Precursor Protein (pf10515); CAP-Gly: Cytoskeletal-Associated protein (pf00225); HELP: Hydrophibic EMAP-Like Protein (pf03451); WD40: β-transducin repeat (pf00400); SiP: Signal peptide; Ig: Immunoglobulin-like domain (pf00047); FN3: Fibronectin type III domain (pf00041); AT-hook: DNA-binding for A/T-rich regions (pf02178); PHD finger: Plant HomeoDomain (Cys)4-His-(Cys)3 (pf00628). Gene names are labelled with a ¹ if exclusion of the alternatively splice regions directly affects structural domains. Note all ASEs are multiples of 3 nucleotides, thus all the alternative splicing events confer in frame peptide omission or insertion. <b>b. Brain-specific alternative splicing is conserved in vertebrates, and possibly beyond, in microtubule-associated genes.</b> Metazoan genomes in Ensembl were searched for paralogs and orthologs of each target gene and for the presence (yellow) or absence (gray) of potential ASEs (Accession numbers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125998#pone.0125998.s005" target="_blank">S1 Table</a>). Yellow indicates that alternatively spliced mRNAs were detected in EST databases. Orange indicates that there were too few ESTs to conclude. Green indicates the absence of both genomic and EST data. Duplications are indicated by thick lines along with the names of the duplicated genes. An indication of the function of each gene is given on the right.</p