The tissue-specific RNA binding protein T-STAR controls regional splicing patterns of neurexin pre-mRNAs in the brain.

The RNA binding protein T-STAR was created following a gene triplication 520-610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain

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 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

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