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.

A new subtype-specific monoclonal antibody for IAP-survivin identifies high-risk patients with diffuse large B-cell lymphoma and improves the prognostic value of bcl-2.

Anti-apoptotic factors including IAP-survivin and bcl-2 are involved in carcinogenesis and predict for disease outcome for patients with cancer. We used RT-PCR and specific primers to generate two recombinant IAP-survivin proteins; one encoding for the full-length protein and the second comprising the survivin sequence incorporating amino acids 98 to 142. Both proteins were used to immunize mice and as capture antigens to screen NS1/immune splenocyte hybridoma supernatants for anti-survivin antibody in ELISA assays. The antibody designated F2-9C3 was most effective and reacted with both recombinant proteins and with the native protein present in lysates of A549 (lung carcinoma) and Jurkat cells in Western blots, immunoprecipitation and formalin-fixed tissue sections. Immunohistochemical staining of normal and neoplastic tissues showed association of the F2-9C3 antibody with the mitotic spindles. Expression of survivin was not detected elsewhere in sections of normal tissue while all neoplastic tissues examined, including those from patients with diffuse large B-cell lymphoma (DLBCL), showed significant expression of survivin. The intensity and localization of staining in these tumours varied and was observed in cytoplasm and/or nuclei. High nuclear expression of survivin predicted the disease outcome in patients with DLBCL. This association was evident when relating intensity to patient survival (p=0.0321) and strengthened when a score was calculated based on both staining intensity and the proportion of the reactive tumour cells (p=0.0128; reduction in the mean survival times: 35% and 46%, respectively). Elevated expression of bcl-2 protein also identified the high-risk patients (p=0.0095; reduction in mean survival time: 37%). Over-expression of both factors was a more powerful indicator of poor prognosis than either marker alone (p=0.0054, 70% reduction in mean survival time). In conclusion, our novel F2-9C3 monoclonal antibody is effective in determination of expression of IAP-survivin in neoplastic tissue. Nuclear overexpression of IAP-survivin using this antibody predicts the disease outcome in patients with DLBCL and significantly improves the predictive power of bcl-2 in these patients.