Regulation of extreme anoxia tolerance via microRNAs in embryos of the annual killifish Austrofundulus limnaeus

一年生南极鱼的胚胎可以在完全缺氧的情况下存活数月。考虑到胚胎主要由大脑和心脏组织组成，这是特别令人惊讶的，这些组织在哺乳动物中完全依赖氧气的存在来支持它们的新陈代谢。A.胚胎表现出无与伦比的耐缺氧能力。limnaeus使其成为这些研究的极好模型。虽然对代谢休眠的生理学有很多了解，但代谢抑制的主要调节因子和在休眠期间增加对环境胁迫的耐受性的因素迄今尚未发现。本提案中概述的工作使用现代DNA测序技术来解决以下对生物学至关重要的问题：（1）代谢抑制期间细胞过程如何可逆地被阻止（2）线粒体基因组如何控制核基因表达。初步证据表明，小调控RNA分子（srRNA），RNA小于25个核苷酸长，不编码蛋白质，可能在调节支持该物种耐缺氧的细胞过程中发挥关键作用。此外，似乎许多srRNA在线粒体基因组中编码，并具有调节核编码基因表达的潜力。事实上，这些srRNA可能负责协调新陈代谢和细胞增殖的变化，这些变化与进入可逆休眠状态的能力有关，而这种休眠状态是在没有氧气的情况下生存所必需的。Illumina平台上的下一代DNA测序将用于分析和鉴定几乎所有在正常发育和缺氧反应中在胚胎中活跃表达的大分子（蛋白编码信使RNA）和srRNA。该技术将应用于在缺氧耐受性方面差异很大的胚胎，并且还将用于鉴定在常氧和缺氧条件下从胚胎分离的线粒体内驻留的srRNA。将描述常氧和缺氧期间线粒体基因组编码的srRNA的亚细胞定位。生物信息学分析将用于鉴定推定的大RNA靶点，以供小线粒体RNA调节。这些线粒体srRNA赋予缺氧耐受性的能力将通过阻断它们的作用然后筛选缺氧耐受性的变化来在体内测试。这项工作有可能从根本上重新定义线粒体基因组在调节基本细胞应激反应中的作用。细胞来源的小RNA可以控制核基因表达的概念可能会改变我们对基础真核细胞生物学的理解。以前从未有一个直接的机制，线粒体控制核基因表达的概述，尽管许多情况下，在线粒体基因组中的突变与核基因表达的变化，已知与疾病有关。首先，它将有助于培养一支熟练的工作队伍，能够将生物学和生物信息学的现代工具应用于有趣的非模型系统。将现代分子和基因组技术的力量应用于对生物学具有根本重要性的问题，并利用生物体形式和功能的惊人多样性的能力将改变我们对生物学的理解。其次，这项工作有可能导致治疗人类和其他哺乳动物心脏病发作和中风的新疗法。第三，A. Limnaeus胚胎为生态学和进化发育生物学实验室提供了极好的模型。 PI将继续使用这些胚胎开发本科教学实验室项目，以说明环境变化对发育结果的重要性。 这些实验室及其结果将通过互联网向公众提供。最后，PI将继续与美国刀鱼协会一起参与外展教育工作，该协会是一个由数千名成员组成的团体，他们对地球仪的刀鱼生物学和保护感兴趣。

Embryos of the annual killifish Austrofundulus limnaeus can survive for months in the complete absence of oxygen (anoxia). This is especially amazing considering that the embryos are composed primarily of brain and heart tissues, tissues that in mammals are known to rely exclusively on the presence of oxygen to support their metabolism. The unparalleled tolerance of anoxia exhibited by embryos of A. limnaeus makes it an excellent model for these studies. While a great deal is understood concerning the physiology of metabolic dormancy, the master regulators of metabolic depression and factors that increase tolerance of environmental stress during dormancy have to date eluded discovery. The work outlined in this proposal uses modern DNA sequencing techniques to address the following questions of fundamental importance to biology: (1) how are cellular processes reversibly arrested during metabolic depression (2) how can the mitochondrial genome control nuclear gene expression. Preliminary evidence suggests that small regulatory RNA molecules (srRNAs), RNAs less than 25 nucleotides long that do not encode for proteins, may play a critical role in regulating the cellular processes that support tolerance to anoxia in this species. Further, it appears that a number of srRNAs are encoded in the mitochondrial genome and have the potential to regulate the expression of nuclear-encoded genes. In fact, these srRNAs may be responsible for orchestrating the changes in metabolism and cell proliferation associated with the ability to enter into a reversible state of dormancy that is required to survive without oxygen. Next-generation DNA sequencing on the Illumina platform will be used to profile and identify nearly all large (protein coding messenger RNAs) and srRNAs that are actively expressed in embryos during normal development and in response to anoxia. This technique will be applied to embryos that differ substantially in their tolerance of anoxia, and will also be used to identify srRNAs resident within mitochondria isolated from embryos under normoxic and anoxic conditions. The subcellular localization of srRNAs encoded by the mitochondrial genome during normoxia and anoxia will be described. Bioinformatics analysis will be used to identify putative large RNA targets for regulation by small mitochondrial RNAs. The ability of these mitochondrial srRNAs to confer tolerance of anoxia will be tested in vivo by blocking their action and then screening for changes in tolerance to anoxia. This work has the potential to fundamentally redefine the role of the mitochondrial genome in regulating the basic cellular stress response. The concept that mitochondrially-derived small RNAs can control nuclear gene expression could transform our understanding of basic eukaryotic cell biology. Never before has a direct mechanism for mitochondrial control of nuclear gene expression been outlined, despite many instances associated with diseases where mutations in the mitochondrial genome are known to be associated with changes in nuclear gene expression.The broader impacts of this work are manifold. First, it will contribute to the training of a skilled work force that can apply modern tools of biology and bioinformatics to interesting nonmodel systems. The ability to apply the power of modern molecular and genomic-scale techniques to questions of fundamental importance to biology and capitalize on the amazing diversity of organismal form and function is going to transform our understanding of biology. Second, this work has the potential to lead to novel therapies for the treatment of heart attack and stroke in humans and other mammals. Third, A. limnaeus embryos provide an excellent model for ecological and evolutionary developmental biology labs. The PI will continue to develop undergraduate teaching lab projects using these embryos to illustrate the importance of environmental variation on developmental outcomes. These labs and their results will be made available to the general public via the internet. Finally, The PI will continue to participate in outreach educational efforts with the American Killifish Association, a group of several thousand members that are interested in killifish biology and conservation across the globe.