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• 1. [Article] Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i>

  Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although ...

  Citation × Citation Citation Abstract Copy Title: Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i> Author: Kacharia, Fenil Rashmin Year: 2016 Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria. Title: Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i> Author: Kacharia, Fenil Rashmin Year: 2016 Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria. Close

• 2. [Article] Genome Rearrangements Can Make and Break Small RNA Genes

  Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are ...

  Citation × Citation Citation Abstract Copy Title: Genome Rearrangements Can Make and Break Small RNA Genes Author: Raghavan, Rahul, Kacharia, Fenil R., Millar, Jess A, Sislak, Christine Demko, Ochman, Howard Year: 2015 Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are conserved among enteric bacteria, but because of the regulatory roles of sRNAs, differences in sRNA repertoires might be responsible for features that differentiate closely related species. We scanned the E. coli MG1655 and Salmonella enterica Typhimurium genomes for nonsyntenic IGRs as a potential source of uncharacterized, species-specific sRNAs and found that genome rearrangements have reconfigured several IGRs causing the disruption and formation of sRNAs. Within an IGR that is present in E. coli but was disrupted in Salmonella by a translocation event is an sRNA that is associated with the FNR/CRP global regulators and influences E. coli biofilm formation. A Salmonella-specific sRNA evolved de novo through point mutations that generated a σ70 promoter sequence in an IGR that arose through genome rearrangement events. The differences in the sRNA pools among bacterial species have previously been ascribed to duplication, deletion, or horizontal acquisition. Here, we show that genomic rearrangements also contribute to this process by either disrupting sRNA-containing IGRs or creating IGRs in which novel sRNAs may evolve. Title: Genome Rearrangements Can Make and Break Small RNA Genes Author: Raghavan, Rahul, Kacharia, Fenil R., Millar, Jess A, Sislak, Christine Demko, Ochman, Howard Year: 2015 Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are conserved among enteric bacteria, but because of the regulatory roles of sRNAs, differences in sRNA repertoires might be responsible for features that differentiate closely related species. We scanned the E. coli MG1655 and Salmonella enterica Typhimurium genomes for nonsyntenic IGRs as a potential source of uncharacterized, species-specific sRNAs and found that genome rearrangements have reconfigured several IGRs causing the disruption and formation of sRNAs. Within an IGR that is present in E. coli but was disrupted in Salmonella by a translocation event is an sRNA that is associated with the FNR/CRP global regulators and influences E. coli biofilm formation. A Salmonella-specific sRNA evolved de novo through point mutations that generated a σ70 promoter sequence in an IGR that arose through genome rearrangement events. The differences in the sRNA pools among bacterial species have previously been ascribed to duplication, deletion, or horizontal acquisition. Here, we show that genomic rearrangements also contribute to this process by either disrupting sRNA-containing IGRs or creating IGRs in which novel sRNAs may evolve. Close

• 3. [Article] Developmental Mechanisms that Support Genome Stability and Embryonic Survival in Stress-tolerant Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i>

  In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. ...

  Citation × Citation Citation Abstract Copy Title: Developmental Mechanisms that Support Genome Stability and Embryonic Survival in Stress-tolerant Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Wagner, Josiah Tad Year: 2015 In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. The annual killifish Austrofundulus limnaeus is possibly the most well described extremophile vertebrate and its embryos have been shown to tolerate extremes in oxygen, salinity, and water availability. This phenotype is likely a result of the annual killifish life history, which includes periods of temporary habitat desiccation and oxygen deprivation, and requires the production of stress-tolerant embryos that depress metabolism in a state of suspended animation, known as diapause. Over the past several decades, the basic morphology and physiology of annual killifish development has become better characterized. However, there are still basic cellular processes that remain to be described in annual killifish such as A. limnaeus. Specifically, changes in DNA structure, expression, and copy number are known to have profound impacts on the phenotype and survival of an organism. Little is known as to how A. limnaeus maintains genome integrity during cell stress, nor how the A. limnaeus nuclear and mitochondrial genomes may have evolved under the unpredictable conditions in which A. limnaeus thrive. Early annual killifish embryonic development is also characterized by a complete dispersion and subsequent reaggregation of embryonic blastomeres prior to formation of the embryonic axis. This unusual period of early development may provide a functional adaptation that allows annual killifish embryos to survive these extreme conditions. The overall goals of this project were to (1) characterize the ability of A. limnaeus to tolerate and repair DNA damage through enzymatic and developmental mechanisms, (2) to determine possible consequences of mitochondrial DNA sequence and copy number on the metabolism of A. limnaeus, and (3) to establish a draft genome of A. limnaeus for future comparative genome studies. The results of this project show that embryos of A. limnaeus have an impressive ability to survive and reverse high doses of DNA damage induced by ultraviolet-C (UV-C) radiation, especially when allowed to recover under photoreactivating light. Surprisingly, embryos that survived irradiation during blastomere dispersion phases were able to develop normally. Characterization of gene expression during embryonic development for genes important for axis formation and cellular differentiation suggests that A. limnaeus embryos may delay axis formation until several days after epiboly is complete, thus allowing time for cells that become damaged to be replaced by surrounding pluripotent cells. This outcome would represent first case of a developmental buffering stage in a vertebrate. A. limnaeus embryos are also unique in their mitochondrial response to anoxia. Whereas in other species the amount of mitochondrial DNA (mtDNA) copy number fluctuates following extremes in oxygen availability, A. limnaeus embryonic mtDNA remains stable. Additionally, characterization of the fully sequenced A. limnaeus mitochondrial genome reveals possible evolutionary adaptations that may have facilitated dormancy and anoxia tolerance when compared to other species within the Order Cyprinodontiformes. The final chapter of this project characterizes the draft genome of A. limnaeus and I provide evidence suggesting that epigenetic DNA methylation that may be involved in regulating diapause. Title: Developmental Mechanisms that Support Genome Stability and Embryonic Survival in Stress-tolerant Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Wagner, Josiah Tad Year: 2015 In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. The annual killifish Austrofundulus limnaeus is possibly the most well described extremophile vertebrate and its embryos have been shown to tolerate extremes in oxygen, salinity, and water availability. This phenotype is likely a result of the annual killifish life history, which includes periods of temporary habitat desiccation and oxygen deprivation, and requires the production of stress-tolerant embryos that depress metabolism in a state of suspended animation, known as diapause. Over the past several decades, the basic morphology and physiology of annual killifish development has become better characterized. However, there are still basic cellular processes that remain to be described in annual killifish such as A. limnaeus. Specifically, changes in DNA structure, expression, and copy number are known to have profound impacts on the phenotype and survival of an organism. Little is known as to how A. limnaeus maintains genome integrity during cell stress, nor how the A. limnaeus nuclear and mitochondrial genomes may have evolved under the unpredictable conditions in which A. limnaeus thrive. Early annual killifish embryonic development is also characterized by a complete dispersion and subsequent reaggregation of embryonic blastomeres prior to formation of the embryonic axis. This unusual period of early development may provide a functional adaptation that allows annual killifish embryos to survive these extreme conditions. The overall goals of this project were to (1) characterize the ability of A. limnaeus to tolerate and repair DNA damage through enzymatic and developmental mechanisms, (2) to determine possible consequences of mitochondrial DNA sequence and copy number on the metabolism of A. limnaeus, and (3) to establish a draft genome of A. limnaeus for future comparative genome studies. The results of this project show that embryos of A. limnaeus have an impressive ability to survive and reverse high doses of DNA damage induced by ultraviolet-C (UV-C) radiation, especially when allowed to recover under photoreactivating light. Surprisingly, embryos that survived irradiation during blastomere dispersion phases were able to develop normally. Characterization of gene expression during embryonic development for genes important for axis formation and cellular differentiation suggests that A. limnaeus embryos may delay axis formation until several days after epiboly is complete, thus allowing time for cells that become damaged to be replaced by surrounding pluripotent cells. This outcome would represent first case of a developmental buffering stage in a vertebrate. A. limnaeus embryos are also unique in their mitochondrial response to anoxia. Whereas in other species the amount of mitochondrial DNA (mtDNA) copy number fluctuates following extremes in oxygen availability, A. limnaeus embryonic mtDNA remains stable. Additionally, characterization of the fully sequenced A. limnaeus mitochondrial genome reveals possible evolutionary adaptations that may have facilitated dormancy and anoxia tolerance when compared to other species within the Order Cyprinodontiformes. The final chapter of this project characterizes the draft genome of A. limnaeus and I provide evidence suggesting that epigenetic DNA methylation that may be involved in regulating diapause. Close