👤 Gavin J Clowry

Neurexins (NRXNs) are presynaptic terminal proteins and candidate neurodevelopmental disorder susceptibility genes; mutations presumably upset synaptic stabilization and function. However, analysis of human cortical tissue samples by RNAseq and quantitative real-time PCR at 8-12 postconceptional weeks, prior to extensive synapse formation, showed expression of all three NRXNs as well as several potential binding partners. However, the levels of expression were not identical; NRXN1 increased with age and NRXN2 levels were consistently higher than for NRXN3. Immunohistochemistry for each NRXN also revealed different expression patterns at this stage of development. NRXN1 and NRXN3 immunoreactivity was generally strongest in the cortical plate and increased in the ventricular zone with age, but was weak in the synaptogenic presubplate (pSP) and marginal zone. On the other hand, NRXN2 colocalized with synaptophysin in neurites of the pSP, but especially with GAP43 and CASK in growing axons of the intermediate zone. Alternative splicing modifies the role of NRXNs and we found evidence by RNAseq for exon skipping at splice site 4 and concomitant expression of KHDBRS proteins which control this splicing. NRXN2 may play a part in early cortical synaptogenesis, but NRXNs could have diverse roles in development including axon guidance, and intercellular communication between proliferating cells and/or migrating neurons. Show less

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. Show less