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.
Acute brain injury is a leading cause of death and disability. Those who survive are often troubled by lifelong social, cognitive, and behavioral deficits. Many interventions have been tested in clinical trials but few have succeeded. Thus, there is considerable room for improvement. Our proposal tests a novel therapy in CNS injury. Utilizing a traumatic brain injury (TBI) model to induce neuronal death, we will test if inhibition of the novel splicing factor RNA Binding Motif 5 (RBM5), by anthraquinone-2-sulfonic acid (AQ2S), is neuroprotective.
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|Kochanek, Patrick M; Jackson, Travis C; Ferguson, Nikki Miller et al. (2015) Emerging therapies in traumatic brain injury. Semin Neurol 35:83-100|
|Jackson, Travis C; Du, Lina; Janesko-Feldman, Keri et al. (2015) The nuclear splicing factor RNA binding motif 5 promotes caspase activation in human neuronal cells, and increases after traumatic brain injury in mice. J Cereb Blood Flow Metab 35:655-66|