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

Portland State University, Portland OR

Investigators

Abstract

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.