TRA2B is a serine-arginine-rich splicing factor that contributes to the alternative splicing of exons and depletion of Tra2b in the mouse causes early embryonic lethality. It modulates splice site selection in a concentration dependent fashion and associates to target exons either directly via GAA binding motifs or indirectly via interactions with other splice factors. TRA2B is highest expressed in neuronal tissue and testis and its expression and activation are controlled via an autoregulatory self-splicing mechanism and by phosphorylation. Depending on interactors and on the position of binding, TRA2B can promote either inclusion or skipping of exons. TRA2B has been associated with splicing processes involved in development, vascularization, spermatogenesis and neuronal function and deregulation of splicing processes has been linked to conditions like Alzheimer’s disease, dementia and Parkinson’s disease. Importantly, TRA2B is involved in the splicing of the SMN transcript and has been demonstrated to promote inclusion of exon 7. The functional loss of the SMN1 gene causes spinal muscular atrophy, whereas the copy number of SMN2, which is alternatively spliced to mainly block exon 7 inclusion, is the major determinant of the disease severity. Correction of the SMN2 splicing pattern to increase the number of functional full-length transcripts is a promising avenue for SMA therapy. The use of HDAC inhibitors like VPA has been demonstrated to correct the SMN2 splicing pattern to a great extent by transcriptional upregulation of TRA2B. Despite promising trial of VPA in SMA therapy and successful application in other conditions like epilepsy and bipolar disorder, it can be assumed that the splicing of other transcripts targeted by TRA2B besides SMN2 will be affected by its upregulation.
In the present work a neuronal-specific Tra2b knock-out mouse was generated to identify transcripts targeted by Tra2b in the central nervous system. Mice homozygously depleted of Tra2b in the central nervous system died shortly after birth and presented severe abnormalities in cortical development. These included a loss of cortical patterning, a reduction of the total cortical width and ventriculomegaly. By immunohistochemical analyses of brain sections at developmental stages reaching from 14.5 dpc to birth, massive apoptosis was detected in the ventricular and subventricular layers of the lateral ventricles and in the thalamic region. Apoptosis at 14.5 dpc was followed by a loss of the proliferative potential in a timely fashion, which caused a progressive loss of cortical material and ventricular dilation that reached its end stage at 16-17 dpc. Based on a mosaic knock-out of Tra2b in the murine brain, mRNA and protein levels of Tra2b were drastically reduced, but not fully absent as demonstrated by quantitative PCR and semi-quantitative Western Blot. Heterozygous knock-out animals were fully viable, showed no apoptosis, normal brain development and Tra2b protein levels comparable to control mice. Analysis of Tra2b isoforms revealed that the autoregulatory splicing feedback-loop of Tra2b is functional in vivo in the mouse brain and compensates for the loss of a single gene copy by upregulation of functional Tra2b transcripts due to skipping of exon 2.
By the use of quantitative PCR, previously and in minigene approaches identified Tra2b-dependent splicing processes of Mapt, Cltb, Tra2a and Nasp were indentified in vivo providing proof of concept for the identification of splicing processes in the Tra2b-deficient murine brain. For the in vivo identification of novel Tra2b target exons, whole transcriptome sequencing and mouse exon array analysis were performed using whole brain RNA from neuronal-specific knock-out mice. Based on dramatic differences between control and knock-out brains and drastically altered cellular compositions, the reliable identification of targeted exons proved to be difficult. Exons of the Shugoshin-like 2 and Tubulin delta chain were identified via exon array analysis to be promoted by Tra2b in vivo. These splicing processes were confirmed by quantitative PCR and both identified exons were shown to be responsive to increased Tra2b concentrations in a minigene splicing assay. Some of the in vivo identified Tra2b-dependent splicing processes have possible implications in neuronal development and function, and might therefore contribute to the observed aberrations in brain development.
Moreover, the cell cycle regulating gene Cdkn1a (encoding p21) was found upregulated in brains of neuronal-specific knock-out mice using exon arrays, and p21 upregulation was validated by quantitative PCR. P21 is a well-known inhibitor of cell cycle progression that causes cells to enter cell cycle arrest at the G1-S-phase transition. RNAi-mediated depletion of Tra2b in NSC34 neuronal precursor cells caused a drastic increase of p21 expression on RNA and on protein level. Strikingly, NSC34 cells died shortly after p21 expression had increased, indicating an apoptotic effect. This suggests that an increased p21 expression is likely the reason for apoptosis and the loss of the proliferative potential in neurogenic areas of the developing Tra2b-depleted brains. Previous studies have linked deficiency of the histone chaperone Nasp to upregulation of p21 and to apoptosis. Further investigations need to clarify, whether Tra2b-related missplicing of Nasp (tNasp depletion) is the underlying reason for p21 upregulation and apoptosis
