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- All Subjects: Genetics
- Creators: Mangone, Marco
Description
Vertebrate genomes demonstrate a remarkable range of sizes from 0.3 to 133 gigabase pairs. The proliferation of repeat elements are a major genomic expansion. In particular, long interspersed nuclear elements (LINES) are autonomous retrotransposons that have the ability to "cut and paste" themselves into a host genome through a mechanism called target-primed reverse transcription. LINES have been called "junk DNA," "viral DNA," and "selfish" DNA, and were once thought to be parasitic elements. However, LINES, which diversified before the emergence of many early vertebrates, has strongly shaped the evolution of eukaryotic genomes. This thesis will evaluate LINE abundance, diversity and activity in four anole lizards. An intrageneric analysis will be conducted using comparative phylogenetics and bioinformatics. Comparisons within the Anolis genus, which derives from a single lineage of an adaptive radiation, will be conducted to explore the relationship between LINE retrotransposon activity and causal changes in genomic size and composition.
ContributorsMay, Catherine (Author) / Kusumi, Kenro (Thesis advisor) / Gadau, Juergen (Committee member) / Rawls, Jeffery A (Committee member) / Arizona State University (Publisher)
Created2013
Description
Gene-centric theories of evolution by natural selection have been popularized and remain generally accepted in both scientific and public paradigms. While gene-centrism is certainly parsimonious, its explanations fall short of describing two patterns of evolutionary and social phenomena: the evolution of sex and the evolution of social altruism. I review and analyze current theories on the evolution of sex. I then introduce the conflict presented to gene-centric evolution by social phenomena such as altruism and caste sterility in eusocial insects. I review gene-centric models of inclusive fitness and kin selection proposed by Hamilton and Maynard Smith. Based their assumptions, that relatedness should be equal between sterile workers and reproductives, I present several empirical examples that conflict with their models. Following that, I introduce a unique system of genetic caste determination (GCD) observed in hybrid populations of two sister-species of seed harvester ants, Pogonomyrmex rugosus and Pogonomyrmex barbatus. I review the evidence for GCD in those species, followed by a critique of the current gene-centric models used to explain it. In chapter two I present my own theoretical model that is both simple and extricable in nature to explain the origin, evolution, and maintenance of GCD in Pogonomyrmex. Furthermore, I use that model to fill in the gaps left behind by the contributing authors of the other GCD models. As both populations in my study system formed from inter-specific hybridization, I review modern discussions of heterosis (also called hybrid vigor) and use those to help explain the ecological competitiveness of GCD. I empirically address the inbreeding depression the lineages of GCD must overcome in order to remain ecologically stable, demonstrating that as a result of their unique system of caste determination, GCD lineages have elevated recombination frequencies. I summarize and conclude with an argument for why GCD evolved under selective mechanisms which cannot be considered gene-centric, providing evidence that natural selection can effectively operate on non-heritable genotypes appearing in groups and other social contexts.
ContributorsJacobson, Neal (Author) / Gadau, Juergen (Thesis advisor) / Laubichler, Manfred (Committee member) / Pratt, Stephen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disease characterized by progressive muscle loss and weakness. This disease arises from a mutation that occurs on a gene that encodes for dystrophin, which results in observable muscle death and inflammation; however, the genetic changes that result from dystrophin's dysfunctionality remain unknown. Current DMD research uses mdx mice as a model, and while very useful, does not allow the study of cell-autonomous transcriptome changes during the progression of DMD due to the strong inflammatory response, perhaps hiding important therapeutic targets. C. elegans, which has a very weak inflammatory response compared to mdx mice and humans, has been used in the past to study DMD with some success. The worm ortholog of the dystrophin gene has been identified as dys-1 since its mutation phenocopies the progression of the disease and a portion of the human dystrophin gene alleviates symptoms. Importantly, the extracted RNA transcriptome from dys-1 worms showed significant change in gene expression, which needs to be further investigated with the development of a more robust model. Our lab previously published a method to isolate high-quality muscle-specific RNA from worms, which could be used to study such changes at higher resolution. We crossed the dys-1 worms with our muscle-specific strain and demonstrated that the chimeric strain exhibits similar behavioral symptoms as DMD patients as characterized by a shortened lifespan, difficulty in movement, and a decrease in speed. The presence of dys-1 and other members of the dystrophin complex in the body muscle were supported by the development of a resulting phenotype due to RNAi knockdown of each component in the body muscle; however, further experimentation is needed to reinforce this conclusion. Thus, the constructed chimeric C. elegans strain possesses unique characteristics that will allow the study of genetic changes, such as transcriptome rearrangements and dysregulation of miRNA, and how they affect the progression of DMD.
ContributorsNguyen, Thuy-Duyen Cao (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Duchaine, Thomas (Committee member) / School of Social Transformation (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Alternative polyadenylation (APA) is the biological mechanism in which the same gene can have multiple 3'untranslated region (3'UTR) isoforms due to the presence of multiple polyadenylation signal (PAS) elements within the pre mRNAs. Because APA produces mRNA transcripts that have different 3'UTR isoforms, certain transcripts may be subject to post-transcriptional regulation by regulatory non-coding RNAs, such as microRNAs or RNA binding proteins defects of which have been implicated in diseases such as cancer. Despite the increasing level of information, functional understanding of the molecular mechanisms involved in transcription is still poorly understood, nor is it clear why APA is necessary at a cell or tissue-specific level. To address these questions I wanted to develop a set of sensor strain plasmids capable of detecting cleavage and polyadenylation in vivo, inject the complete sensor strain plasmid into C. elegans and prepare stable transgenic lines, and perform proof-of-principle RNAi feeding experiments targeting genes associated with the cleavage and polyadenylation complex machinery. I demonstrated that it was possible to create a plasmid capable of detecting cleavage and polyadenylation in C. elegans; however, issues arose during the RNAi assays indicating the sensor strain plasmid was not sensitive enough to the RNAi to effectively detect in the worms. Once the problems involved with sensitivity and variability in the RNAi effects are resolved, the plasmid would be able to better address questions regarding the functional understanding of molecular mechanisms involved in transcription termination.
ContributorsWilky, Henry Patrick (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Blazie, Stephen (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
MicroRNAs (miRNAs) are 17-22 nucleotide non-coding RNAs that regulate gene expression by targeting non-complementary elements in the 3’ untranslated regions (3’UTRs) of mRNAs. miRNAs, which form complex networks of interaction that differ by tissue and developmental stage, display conservation in their function across metazoan species. Yet much remains unknown regarding their biogenesis, localization, strand selection, and their absolute abundance due to the difficulty of detecting and amplifying such small molecules. Here, I used an updated HT qPCR-based methodology to follow miRNA expression of 5p and 3p strands for all 190 C. elegans miRNAs described in miRBase throughout all six developmental stages in triplicates (total of 9,708 experiments), and studied their expression levels, tissue localization, and the rules underlying miRNA strand selection. My study validated previous findings and identified novel, conserved patterns of miRNA strand expression throughout C. elegans development, which at times correlate with previously observed developmental phenotypes. Additionally, my results highlighted novel structural principles underlying strand selection, which can be applied to higher metazoans.
Though optimized for use in C. elegans, this method can be easily adapted to other eukaryotic systems, allowing for more scalable quantitative investigation of miRNA biology and/or miRNA diagnostics.
ContributorsMeadows, Dalton Alexander (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Murugan, Vel (Committee member) / Wilson-Rawls, Jeanne (Committee member) / Arizona State University (Publisher)
Created2023
Description
Most protein-coding mRNAs in eukaryotes must undergo a series of processing steps so they can be exported from the nucleus and translated into protein. Cleavage and polyadenylation are vital steps in this maturation process. Improper cleavage and polyadenylation results in variation in the 3′ UTR length of genes, which is a hallmark of various human diseases. Previous data have shown that the majority of 3’UTRs of mRNAs from the nematode Caenorhabditis elegans terminate at an adenosine nucleotide, and that mutating this adenosine disrupts the cleavage reaction. It is unclear if the adenosine is included in the mature mRNA transcript or if it is cleaved off. To address this question, we are developing a novel method called the Terminal Adenosine Methylation (TAM) assay which will allow us to precisely define whether the cleavage reaction takes place upstream or downstream of this terminal adenosine. The TAM Assay utilizes the ability of the methyltransferase domain (MTD) of the human methyltransferase METTL16 to methylate the terminal adenosine of a test mRNA transcript prior to the cleavage reaction in vivo. The presence or absence of methylation at the terminal adenosine will then be identified using direct RNA sequencing. This project focuses on 1) preparing the chimeric construct that positions the MTD on the mRNA cleavage site of a test mRNA transcript, and 2) testing the functionality of this construct in vitro and developing a transgenic C. elegans strain expressing it. The TAM assay has the potential to be a valuable tool for elucidating the role of the terminal adenosine in cleavage and polyadenylation.
ContributorsKeane, Sara (Author) / Mangone, Marco (Thesis director) / Lapinaite, Audrone (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of English (Contributor)
Created2023-05
Description
The ability to tolerate bouts of oxygen deprivation varies tremendously across the animal kingdom. Adult humans from different regions show large variation in tolerance to hypoxia; additionally, it is widely known that neonatal mammals are much more tolerant to anoxia than their adult counterparts, including in humans. Drosophila melanogaster are very anoxia-tolerant relative to mammals, with adults able to survive 12 h of anoxia, and represent a well-suited model for studying anoxia tolerance. Drosophila live in rotting, fermenting media and a result are more likely to experience environmental hypoxia; therefore, they could be expected to be more tolerant of anoxia than adults. However, adults have the capacity to survive anoxic exposure times ~8 times longer than larvae. This dissertation focuses on understanding the mechanisms responsible for variation in survival from anoxic exposure in the genetic model organism, Drosophila melanogaster, focused in particular on effects of developmental stage (larval vs. adults) and within-population variation among individuals.
Vertebrate studies suggest that surviving anoxia requires the maintenance of ATP despite the loss of aerobic metabolism in a manner that prevents a disruption of ionic homeostasis. Instead, the abilities to maintain a hypometabolic state with low ATP and tolerate large disturbances in ionic status appear to contribute to the higher anoxia tolerance of adults. Furthermore, metabolomics experiments support this notion by showing that larvae had higher metabolic rates during the initial 30 min of anoxia and that protective metabolites were upregulated in adults but not larvae. Lastly, I investigated the genetic variation in anoxia tolerance using a genome wide association study (GWAS) to identify target genes associated with anoxia tolerance. Results from the GWAS also suggest mechanisms related to protection from ionic and oxidative stress, in addition to a protective role for immune function.
Vertebrate studies suggest that surviving anoxia requires the maintenance of ATP despite the loss of aerobic metabolism in a manner that prevents a disruption of ionic homeostasis. Instead, the abilities to maintain a hypometabolic state with low ATP and tolerate large disturbances in ionic status appear to contribute to the higher anoxia tolerance of adults. Furthermore, metabolomics experiments support this notion by showing that larvae had higher metabolic rates during the initial 30 min of anoxia and that protective metabolites were upregulated in adults but not larvae. Lastly, I investigated the genetic variation in anoxia tolerance using a genome wide association study (GWAS) to identify target genes associated with anoxia tolerance. Results from the GWAS also suggest mechanisms related to protection from ionic and oxidative stress, in addition to a protective role for immune function.
ContributorsCampbell, Jacob B (Author) / Harrison, Jon F. (Thesis advisor) / Gadau, Juergen (Committee member) / Call, Gerald B (Committee member) / Sweazea, Karen L (Committee member) / Rosenberg, Michael S. (Committee member) / Arizona State University (Publisher)
Created2018
Description
Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
ContributorsKotagama, Kasuen Indrajith Bandara (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Newbern, Jason (Committee member) / Rawls, Alan (Committee member) / Arizona State University (Publisher)
Created2019
Description
MicroRNAs (miRNAs) are short non-coding RNAs that play key roles during metazoan development, and are frequently misregulated in human disease. MiRNAs regulate gene output by targeting degenerate elements primarily in the 3´ untranslated regions of mRNAs. MiRNAs are often deeply conserved, but have undergone drastic expansions in higher metazoans, leading to families of miRNAs with highly similar sequences. The evolutionary advantage of maintaining multiple copies of duplicated miRNAs is not well understood, nor has the distinct functions of miRNA family members been systematically studied. Furthermore, the unbiased and high-throughput discovery of targets remains a major challenge, yet is required to understand the biological function of a given miRNA.
I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale.
In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale.
In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
ContributorsWolter, Justin M (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Kusumi, Kenro (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2016
Description
Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by out-of-frame mutations in the dystrophin gene, and the absence of a functional dystrophin protein ultimately leads to instability of the sarcolemma, skeletal muscle necrosis, and atrophy. While the structural changes that occur in dystrophic muscle are well characterized, resulting changes in muscle-specific gene expression that take place in dystrophin’s absence remain largely uncharacterized, as they are potentially obscured by the characteristic chronic inflammation in dystrophin deficient muscle.
The conservation of the dystrophin gene across metazoans suggests that both vertebrate and invertebrate model systems can provide valuable contributions to the understanding of DMD initiation and progression. Specifically, the invertebrate C. elegans possesses a dystrophin protein ortholog, dys-1, and a mild inflammatory response that is inactive in the muscle, allowing for the characterization of transcriptome rearrangements affecting disease progression independently of inflammation. Furthermore, C. elegans do not possess a satellite cell equivalent, meaning muscle regeneration does not occur. This makes C. elegans unique in that they allow for the study of dystrophin deficiencies without muscle regeneration that may obscure detection of subtle but consequential changes in gene expression.
I hypothesize that gaining a comprehensive definition of both the structural and signaling roles of dystrophin in C. elegans will improve the community’s understanding of the progression of DMD as a whole. To address this hypothesis, I have performed a phylogenetic analysis on the conservation of each member of the dystrophin associated protein complex (DAPC) across 10 species, established an in vivo system to identify muscle-specific changes in gene expression in the dystrophin-deficient C. elegans, and performed a functional analysis to test the biological significance of changes in gene expression identified in my sequencing results. The results from this study indicate that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract disease progression. Furthermore, these findings allow for the identification of transcriptome changes that potentially serve as both independent drivers of disease and potential therapeutic targets for the treatment of DMD.
The conservation of the dystrophin gene across metazoans suggests that both vertebrate and invertebrate model systems can provide valuable contributions to the understanding of DMD initiation and progression. Specifically, the invertebrate C. elegans possesses a dystrophin protein ortholog, dys-1, and a mild inflammatory response that is inactive in the muscle, allowing for the characterization of transcriptome rearrangements affecting disease progression independently of inflammation. Furthermore, C. elegans do not possess a satellite cell equivalent, meaning muscle regeneration does not occur. This makes C. elegans unique in that they allow for the study of dystrophin deficiencies without muscle regeneration that may obscure detection of subtle but consequential changes in gene expression.
I hypothesize that gaining a comprehensive definition of both the structural and signaling roles of dystrophin in C. elegans will improve the community’s understanding of the progression of DMD as a whole. To address this hypothesis, I have performed a phylogenetic analysis on the conservation of each member of the dystrophin associated protein complex (DAPC) across 10 species, established an in vivo system to identify muscle-specific changes in gene expression in the dystrophin-deficient C. elegans, and performed a functional analysis to test the biological significance of changes in gene expression identified in my sequencing results. The results from this study indicate that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract disease progression. Furthermore, these findings allow for the identification of transcriptome changes that potentially serve as both independent drivers of disease and potential therapeutic targets for the treatment of DMD.
ContributorsHrach, Heather (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Newbern, Jason (Committee member) / Rawls, Jeffery (Committee member) / Arizona State University (Publisher)
Created2020