Examining the hibernating brain for temperature-sensitive RNA editing

 描述（由申请人提供）：冬眠哺乳动物对一定程度的脑缺血表现出无与伦比的耐受性，这种脑缺血对包括人类在内的非冬眠动物来说是衰弱或致命的。冬眠期间天然神经保护的分子机制在很大程度上是未知的。三个最新进展：1）在寒冷与温暖生活的章鱼中发现了离子通道的自适应RNA编辑; 2）对改变转录组蛋白质输出的转录后机制的日益重视; 3）数据显示冬眠动物大脑蛋白质组中几乎没有定量差异，这使我们提出，温度敏感的RNA编辑被保留为哺乳动物冬眠动物中的异温适应。具体来说，我们假设腺苷脱氨酶作用于RNA（阿达尔）的温度敏感性RNA编辑改变了冬眠动物大脑中的蛋白质结构和功能，以增强神经保护作用。在双链RNA中，阿达尔活性用肌苷（A至I）取代腺苷; RNA的双链特征随着温度的降低而日益稳定。A至I置换可重新编码氨基酸并改变剪接受体、剪接供体或3'端加工和聚腺苷酸化位点，以显著改变转录组的蛋白质输出。包括离子通道和神经受体在内的脑蛋白是包括哺乳动物在内的几个物种中阿达尔编辑的重要靶标。我们将研究在冬眠阶段具有显著不同活动的三个大脑区域，利用我们独特的地松鼠组织库，该组织库包含来自精确定义的200个样本，不同的生理状态，包括核心体温范围从5°C到37°C的动物。目的利用RNA序列分析技术，研究不同体温冬眠不同阶段的转录组特征。将分析数据以鉴定经历明显的状态特异性RNA编辑的转录物，所述RNA编辑将导致影响蛋白质结构的氨基酸取代或剪接变体。将通过比较cDNA和基因组DNA PCR产物的常规测序色谱图来验证推定的RNA编辑变化。目标二中的第二轮更具体的测序实验将使用靶向方法来选择性地富集具有替代3'末端的转录物的检测。在这个探索/发展项目的最后，我们将大大增强对大脑转录组如何在冬眠动物一年的表型周期中改变的理解。如果在冬眠动物的大脑中检测到RNA编辑，我们也将确定它是否会随着哺乳动物体温的降低而增加。通过确定RNA编辑和其他结构变异的特定靶点，这些实验可能揭示冬眠动物增强神经保护的蛋白质亚型，并为在中风，心脏骤停或创伤引起的脑缺血患者中设计类似的神经保护状态提供关键的第一步。

 DESCRIPTION (provided by applicant): Hibernating mammals exhibit unparalleled tolerance to a degree of cerebral ischemia that is debilitating or fatal to non-hibernators, including humans. The molecular mechanisms underlying natural neuroprotection during hibernation are largely unknown. Three recent advances: 1) the discovery of adaptive RNA editing of ion channels in cold vs warm-living octopus; 2) a growing appreciation of post-transcriptional mechanisms that alter the protein output of the transcriptome; and 3) data revealing few quantitative differences in the brain proteome of hibernators, lead us to propose that temperature-sensitive RNA editing is retained as an adaptation for heterothermy in mammalian hibernators. Specifically, we hypothesize that temperature-sensitive RNA editing by Adenosine Deaminase Acting on RNA (ADAR) alters protein structure and function in hibernator brains to enhance neuroprotection. ADAR activity substitutes adenosine with inosine (A-to-I) in double-stranded RNA; the double-stranded character of RNA is increasingly stabilized as temperature is decreased. A-to-I replacements can re-code amino acids and alter splice acceptor, splice donor, or 3' end processing and polyadenylation sites to significantly alter the protein output of the transcriptome. Brain proteins including ion channels and neuroreceptors are important targets of ADAR editing in several species, including mammals. We will study three brain regions with dramatically different activities across hibernation stages, exploiting our unique ground squirrel tissue bank which contains 200 samples from precisely defined, distinct physiological states, and includes animals with core body temperatures ranging from 5°C to 37°C. Aim One will characterize the transcriptome from multiple stages of hibernation that differ by body temperature using RNA- seq. Data will be analyzed to identify transcripts undergoing apparent state-specific RNA editing that would cause amino acid substitution or splice variants impacting protein structure. Putative RNA editing changes will be validated by comparing conventional sequencing chromatograms from cDNA and genomic DNA PCR products. A second, more specific round of sequencing experiments in Aim Two will use a targeted approach to selectively enrich detection of transcripts with alternative 3' ends. At the end of thi exploratory/developmental project we will have a greatly enhanced understanding of how the brain transcriptome alters across phenotypic cycles of the hibernator's year. If RNA editing is detected in hibernator brain, we also will have determined whether it increases as body temperature decreases in a mammal. By identifying specific targets of RNA editing and other structural variation, these experiments may reveal protein isoforms underlying the hibernator's enhanced neuroprotection and provide a critical first step towards engineering an analogous neuroprotected stated in victims of cerebral ischemia by stroke, cardiac arrest or trauma.