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%的人类基因受到信使核糖核酸的剪接。RNA剪接发生在被称为核剪接体的高度专业化的结构中。剪接体由催化目标内含子/外显子切除的几个关键蛋白和调节这些酶的活性/效率的100‘端S辅助因子组成。剪接体辅助因子指导剪接动力学的许多方面，包括不同蛋白质变体在全身的分布。脑损伤和疾病干扰了拼接。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异表达。不适应蛋白变异体的上调可能会加剧神经元死亡，损害中枢神经系统的恢复。RNA结合基序5(RBM5)是一个剪接辅助因子。它在中枢神经系统和睾丸中高度表达，但在健康或受损的大脑中的功能尚不清楚。RBM5短暂定位于剪接体，在剪接体围绕前mRNA靶标组装的早期。一个锌指结构域(RanBP2-Type)和其他几个RNA结合域，协调其与前mRNA靶标中可剪接的外显子的高度选择性相互作用。在癌细胞中，RBM5调节caspase-2(有利于死亡)和c-flip(有利于生存)基因的剪接变体选择。它促进了外显子9与caspase-2和外显子7与c-flip mRNA的互斥。这分别诱导了caspase2L和c-flipl蛋白的表达。Caspase2L是一种亲死亡剪接变体。C-FLIPL剪接变异体也具有促凋亡功能。相反，抑制RBM5分别导致外显子9/7滞留，并迫使caspase-2S/c-Flips表达。Caspase-2s和c-Flips都是强有力的支持生存的变体。因此，RBM5通过上调支持死亡的剪接变体与支持生存的剪接变体的比率来促进细胞死亡。RBM5还没有在大脑中被研究过。在中枢神经系统中的高表达提示它可能在剪接介导的细胞死亡机制中发挥关键作用。就我们所知，从来没有药物被开发出来专门针对大脑中的亲死亡剪接机制-因此剪接定向治疗的治疗价值完全未知。我们的初步数据显示，RBM5在海马CA3结构中高度丰富--这是一个特别容易受到创伤性脑损伤(TBI)影响的神经元群体。此外，我们还发现，世界上第一个小分子RBM5抑制剂(阻断RanBP2-型结构域)--蒽醌-2-磺酸(AQ2S)在体外和体内都能减少脑损伤后神经元的死亡。这项拨款提案旨在验证我们的假设，即RBM5是大鼠/人神经元中强大的亲死亡剪接因子(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，AQ2S是第一个可行的药物来阻断大脑中的亲死亡剪接机制。

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.