First Advisor

Jason Podrabsky

Date of Publication

Fall 12-27-2017

Document Type


Degree Name

Doctor of Philosophy (Ph.D.) in Biology






Non-coding RNA, Anoxemia, Gene expression, Vertebrates, Killifishes -- Embryos -- Effect of stress on



Physical Description

1 online resource (x, 177 pages)


Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet requires increased expression of specific proteins. Studying the rapid and complex regulation of gene expression associated with survival of anoxia may uncover new mechanisms of cellular biology and transform our understanding of cells, as well as inform prevention and treatment of heart attack and stroke in humans. Small non-coding RNAs (sncRNAs) have emerged as regulators of gene expression that can be rapidly employed, can target individual genes or suites of genes, and are highly conserved across species. There are diverse types of sncRNAs, some coopted from degradation of longer RNAs in the cell. The sncRNA revolution has yielded a large body of literature revealing the roles of sncRNAs in a myriad of biological processes, from development to regulation of the cell cycle and apoptosis, to responding to stress, including freezing, dehydration, ischemia, and anoxia. Given the regulatory complexity required to survive anoxia, examining sncRNAs in the context of extreme anoxia tolerance has the potential to expand our understanding of the role that sncRNAs may play in basic cell biology, as well as in response to stresses such as anoxia. A comparative model including anoxia-tolerant and anoxia-sensitive phenotypes allows us to better identify sncRNAs that likely play a critical role in anoxia tolerance. Embryos of A. limnaeus are the most anoxia tolerant vertebrate known and are comprised of a range of anoxia-tolerance phenotypes. These characteristics create a unique opportunity for comparative study of the role of sncRNAs in anoxia tolerance in phenotypes with a common genomic background. The overall goals of this project were to: (1) describe the sncRNA transcriptome and changes in its expression in response to anoxia in the embryos of A. limnaeus and in other anoxia-tolerant vertebrates, and (2) to identify specific sncRNAs of interest based on these sequencing projects and to follow-up on their biogenesis, localization, and function in A. limnaeus embryos and a continuous cell line derived from A. limnaeus embryos. Chapter 2 focuses on the identity and expression of sncRNAs in embryos of A. limnaeus in 4 embryonic stages that differ in their anoxia tolerance and physiology. Chapter 3 explores sncRNA expression in brain tissue (the most oxygen-sensitive organ) in other anoxia-tolerant vertebrates: the crucian carp, western painted turtle, leopard frog, and epaulette shark. This allows us to assess the similarities and differences in sncRNA biology in species that evolved anoxia independently, and put the findings from A. limnaeus in an evolutionary context. Chapter 4 describes the establishment of the AL4 anoxia-tolerant cell line derived from A. limnaeus embryos, which allows for more detailed study of particular sncRNAs of interest in Chapter 5. Using whole embryos of A. limnaeus and the AL4 cell line, Chapter 5 describes the expression, localization, and possible biogenesis and mechanism of action of mitochondria-derived sncRNAs, known as mitosRNAs. Chapter 6 summarizes the findings and discusses potential future directions. The work in this dissertation represents the first global survey of sncRNA expression in anoxia tolerant vertebrates. While many interesting patterns of expression were identified, the most interesting discovery is the expression of sncRNAs that are generated in the mitochondria, but have the potential to function in other compartments of the cell. This discovery could transform the way we view the role of the mitochondria in regulating gene expression in eukaryotic cells.


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Persistent Identifier

F S2-1.pdf (24056 kB)
Full heatmaps of sncRNAs differentially expressed over development at t=0 (normoxia)

F S2-2.pdf (1637 kB)
Full heatmaps of sncRNAs differentially expressed in response to anoxia within a stage

T S2.1.xlsx (48 kB)
Summarizing information for all samples

T S2.2.xlsx (38 kB)
Known Stress-responsive miRNA database

T S2.4.xlsx (39 kB)
Top 100 most highly expressed sncRNAs at t=0 (normoxia) of each stage

T S2.3.xlsx (26481 kB)
sncRNA catalog lists all unique sncRNAs in study

T S2.5.xlsx (5973 kB)
SncRNAs differentially expressed at t=0 over development

T S2.6.xlsx (1716 kB)
SncRNAs differentially expressed in response to anoxia within a stage

Supplementary methods.pdf (91 kB)
Supplementary methods for ancillary data collected for turtles

T S3.1.xlsx (12 kB)
Ancillary data collected for turtles

T S3.2.xlsx (25 kB)
Sample information for sncRNA samples from anoxia-tolerant vertebrates

T S3.3.xlsx (13294 kB)
Catalog of sncRNAs in anoxia-tolerant vertebrates

T S3.4.xlsx (190 kB)
sncRNAs differentially expressed in response to anoxia and recovery in anoxia-tolerant vertebrates

T S5.1.xlsx (9 kB)
RNA extraction information for cell samples for sncRNA analysis

F S3.1.pdf (91 kB)
RNA yield per gram of brain tissue for each animal during each treatment

F S3.2.pdf (56 kB)
Length distribution of unique small RNAs identified in each species