The mRNA splicing factor RBM5: A new therapeutic target for TBI

描述（由申请人提供）：报告估计超过90%的人类基因受到mRNA剪接。RNA剪接发生在称为核剪接体的高度特化结构中。剪接体由催化靶内含子/外显子切除的几种关键蛋白质以及调节这些酶的活性/效率的100多种辅因子组成。剪接体辅因子指导剪接动力学的许多方面，包括不同蛋白质变体在整个身体中的分布。脑损伤和疾病干扰剪接。剪接体机制的病理变化改变了不同生存和死亡蛋白的剪接变体表达。适应不良蛋白变体的上调可能加剧神经元死亡并损害CNS恢复。 RNA结合基序5（RBM 5）是一种剪接辅因子。它在中枢神经系统和睾丸中高度表达，但其在健康或受损脑中的功能尚不清楚。RBM 5在剪接体组装期间非常早地在前mRNA靶周围短暂定位于剪接体。锌指结构域（RanBP 2型）和其他几个RNA结合结构域，协调其与前体mRNA靶标中的可剪接外显子的高度选择性相互作用。在癌细胞中，RBM 5调节caspase-2（促死亡）和c-FLIP（促存活）基因的剪接变体选择。促进caspase-2的外显子9和c-FLIP mRNA的外显子7的排除。这分别诱导半胱天冬酶2L和c-FLIPL蛋白表达。Caspase 2L是促死亡剪接变体。c-FLIPL剪接变体也具有促凋亡功能。相反，RBM 5抑制分别导致外显子9/7保留，并迫使caspase-2s/c-FLIPs表达。Caspase-2s和c-FLIPs都是有效的促存活变体。因此，RBM 5通过上调促死亡与促存活剪接变体的比率来促进细胞死亡。 RBM 5尚未在大脑中进行研究。在中枢神经系统中的高表达表明它可能在剪接介导的细胞死亡机制中起关键作用。据我们所知，还没有开发出专门针对大脑中促死亡剪接机制的药物-因此剪接定向疗法的治疗价值完全未知。我们的初步数据显示，RBM 5在海马CA 3形成中高度富集-这是一种特别容易受到创伤性脑损伤（TBI）影响的神经元群体。此外，我们发现，蒽醌-2-磺酸（AQ 2S），世界上第一个小分子RBM 5抑制剂（阻断RanBP 2型结构域），减少TBI后的神经元死亡在体外和体内。这项拨款提案旨在验证我们的假设，即RBM 5是大鼠/人类神经元中的一种有效的促死亡剪接因子（即显示它上调这些细胞中的caspase-2L和c-FLIPL剪接），并进一步证实我们的初步数据，表明AQ 2S是第一种阻断大脑中促死亡剪接机制的可行药物。

DESCRIPTION (provided by applicant): Reports estimate that over 90% of all human genes are subject to mRNA splicing. RNA splicing occurs in highly specialized structures termed nuclear spliceosomes. Spliceosomes consist of a few key proteins that catalyze the excision of target introns/exons, plus 100's of co-factors that regulate the activity/efficiency of these enzymes. Spliceosome co-factors direct many facets of splicing dynamics including distribution of different protein variants throughout the body. Brain injury and disease disturb splicing. Pathological changes in spliceosome mechanics alter splice variant expression of different survival and death proteins. An upregulation of maladaptive protein variants may exacerbate neuronal death and impair CNS recovery. RNA Binding Motif 5 (RBM5) is a splicing co-factor. It is highly expressed in the CNS and testis but its function in the healthy or injured brain is unknown. RBM5 briefly localizes to the spliceosome, very early during spliceosome assembly around pre-mRNA targets. A zinc finger domain (RanBP2-Type), and several other RNA binding domains, coordinate its highly selective interaction with spliceable exons in pre-mRNA targets. In cancer cells, RBM5 regulates splice variant selection of caspase-2 (pro-death) and c-FLIP (pro-survival) genes. It promotes the exclusion of exon 9 from caspase-2, and exon 7 from c-FLIP mRNA. This induces caspase2L and c-FLIPL protein expression, respectively. Caspase2L is a pro-death splice variant. The c-FLIPL splice variant also has pro-apoptotic functions. In contrast, RBM5 inhibition causes exon 9/7 retention, respectively, and forces caspase-2s/c-FLIPs expression. Caspase-2s and c-FLIPs are both potent pro-survival variants. Thus RBM5 promotes cell death by upregulating the ratio of pro-death to pro-survival splice variants. RBM5 has not been studied in the brain. High expression in the CNS suggests that it may play a key role in splicing-mediated cell death mechanisms. To the best of our knowledge, no drug has ever been developed to specifically target pro-death splicing mechanisms in the brain - thus the therapeutic value of splicing directed therapies is completely unknown. Our preliminary data show that RBM5 is highly enriched in the hippocampal CA3 formation - a neuron population that is especially vulnerable to traumatic brain injury (TBI). Furthermore, we show that anthraquinone-2-sulfonic acid (AQ2S), the world's first small-molecule RBM5 inhibitor (that blocks the RanBP2-Type domain), decreases neuronal death after TBI in vitro and in vivo. This grant proposal seeks to test our hypothesis that RBM5 is a potent pro-death splicing factor in rat/human neurons (i.e. show that it upregulates caspase-2L and c-FLIPL splicing in these cells), and further confirm our preliminary data showing that AQ2S is the first viable drug to block pro-death splicing mechanisms in the brain.