Neuronal-specific Deficiency of the Splicing Factor Tra2b Causes p21-mediated Apoptosis in Neurogenic Areas of the Developing Mouse Brain

Alternative splicing (AS) increases the informational content of the genome and is more prevalent in the brain than in any other tissue. The splicing factor Tra2b (Sfrs10) can modulate splicing inclusion of exons by specifically detecting GAA-rich binding motifs and its absence causes early embryonic lethality in mice. TRA2B has been shown to be involved in splicing processes of Nasp (nuclear autoantigenic sperm protein), MAPT (microtubule associated protein tau) and SMN (survival motor neuron), and is therefore implicated in spermatogenesis and neurological diseases like Alzheimer's disease, dementia, Parkinson's disease and spinal muscular atrophy. Here we generated a neuronal-specific Tra2b knock-out mouse that lacks Tra2b expression in neuronal and glial precursor cells by using the Nestin-Cre. Neuronal-specific Tra2b knock-out mice die immediately after birth and show severe abnormalities in cortical development, which are caused by massive apoptotic events in the ventricular layers of the cortex, demonstrating a pivotal role of Tra2b for the developing central nervous system. Using whole brain RNA on exon arrays we identified differentially expressed alternative exons of Tubulin delta1 and Shugoshin like2 as in vivo targets of Tra2b. Most interestingly, we found increased expression of the cyclin dependent kinase inhibitor 1a (p21) which we could functionally link to neuronal precursor cells in the affected brain regions. We provide further evidence that the absence of Tra2b causes p21 upregulation and ultimately cell death in NSC34 neuronal-like cells. These findings demonstrate that Tra2b regulates splicing events essential for maintaining neuronal viability during development. Apoptotic events triggered via p21 might not be restricted to the developing brain but could possibly be generalized to the whole organism and explain early embryonic lethality in Tra2b-depleted mice.可变剪接（Alternative Splicing, AS）可提升基因组的信息承载量，且在大脑中的发生频率高于其他所有组织。剪接因子Tra2b（Sfrs10）能够通过特异性识别富含GAA的结合基序，调控外显子的剪接保留；在小鼠体内，该因子的缺失会引发早期胚胎致死。已有研究证实，Tra2b参与Nasp（核自身抗原精子蛋白，nuclear autoantigenic sperm protein）、MAPT（微管相关蛋白tau，microtubule associated protein tau）以及SMN（运动神经元生存蛋白，survival motor neuron）的剪接过程，因此其与精子发生以及阿尔茨海默病、痴呆、帕金森病、脊髓性肌萎缩症等神经系统疾病密切相关。本研究借助巢蛋白-Cre（Nestin-Cre）系统，构建了在神经元及胶质前体细胞中缺失Tra2b表达的神经元特异性Tra2b敲除小鼠。该类敲除小鼠在出生后即刻死亡，并表现出严重的大脑皮层发育异常；该异常由皮层室管膜层发生的大量细胞凋亡事件所导致，这证明Tra2b在中枢神经系统发育过程中发挥关键作用。本研究通过全脑RNA外显子芯片，鉴定出微管蛋白delta1（Tubulin delta1）与Shugoshin样蛋白2（Shugoshin like2）的差异表达可变外显子，作为Tra2b的体内调控靶标。尤为值得关注的是，本研究发现细胞周期蛋白依赖性激酶抑制剂1a（cyclin dependent kinase inhibitor 1a，p21）的表达上调，且该现象可与受影响脑区中的神经元前体细胞建立功能关联。本研究进一步提供证据表明，在NSC34神经元样细胞中，Tra2b的缺失会导致p21表达上调，最终引发细胞死亡。上述研究结果证实，Tra2b可调控发育过程中维持神经元存活所必需的剪接事件。通过p21介导的细胞凋亡事件可能不仅局限于发育中的大脑，还可能广泛存在于整个生物体中，这或许能够解释Tra2b缺失小鼠的早期胚胎致死现象。