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- All Subjects: Genetics
- Creators: Mangone, Marco
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
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
One of the fundamental questions in molecular biology is how genes and the control of their expression give rise to so many diverse phenotypes in nature. The mRNA molecule plays a key role in this process as it directs the spatial and temporal expression of genetic information contained in the DNA molecule to precisely instruct biological processes in living organisms. The region located between the STOP codon and the poly(A)-tail of the mature mRNA, known as the 3′Untranslated Region (3′UTR), is a key modulator of these activities. It contains numerous sequence elements that are targeted by trans-acting factors that dose gene expression, including the repressive small non-coding RNAs, called microRNAs.
Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored.
This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression.
Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored.
This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression.
ContributorsBlazie, Stephen M (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Josh (Committee member) / Lake, Doug (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2016
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 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
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
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
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
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
Schizophrenia, a debilitating neuropsychiatric disorder, affects 1% of the population. This multifaceted disorder is comprised of positive (hallucinations/psychosis), negative (social withdrawal/anhedonia) and cognitive symptoms. While treatments for schizophrenia have advanced over the past few years, high economic burdens are still conferred to society, totaling more than $34 billion in direct annual costs to the United States of America. Thus, a critical need exists to identify the factors that contribute towards the etiology of schizophrenia. This research aimed to determine the interactions between environmental factors and genetics in the etiology of schizophrenia. Specifically, this research shows that the immediate early gene, early growth response 3 (EGR3), which is upregulated in response to neuronal activity, resides at the center of a biological pathway to confer risk for schizophrenia. While schizophrenia-risk proteins including neuregulin 1 (NRG1) and N-methyl-D-aspartate receptors (NMDAR’s) have been identified upstream of EGR3, the downstream targets of EGR3 remain relatively unknown. This research demonstrates that early growth response 3 regulates the expression of the serotonin 2A-receptor (5HT2AR) in the frontal cortex following the physiologic stimulus, sleep deprivation. This effect is translated to the level of protein as 8 hours of sleep-deprivation results in the upregulation of 5HT2ARs, a target of antipsychotic medications. Additional downstream targets were identified following maximal upregulation of EGR3 through electroconvulsive stimulation (ECS). Both brain-derived neurotrophic factor (BDNF) and its epigenetic regulator, growth arrest DNA-damage-inducible 45 beta (GADD45B) are upregulated one-hour following ECS in the hippocampus and require the presence of EGR3. These proteins play important roles in both cellular proliferation and dendritic structural changes. Next, the effects of ECS on downstream neurobiological processes, hippocampal cellular proliferation and dendritic structural changes were examined. Following ECS, hippocampal cellular proliferationwas increased, and dendritic structural changes were observed in both wild-type and early growth response 3 knock-out (Egr3-/-) mice. Effects in the number of dendritic spines and dendritic complexity following ECS were not found to require EGR3. Collectively, these results demonstrate that neuronal activity leads to the regulation of schizophrenia risk proteins by EGR3 and point to a possible molecular mechanism contributing risk for schizophrenia.
ContributorsMeyers, Kimberly (Author) / Gallitano, Amelia L (Thesis advisor) / Newbern, Jason (Thesis advisor) / Mangone, Marco (Committee member) / Nikulina, Ella (Committee member) / Qiu, Shenfeng (Committee member) / Ferguson, Deveroux (Committee member) / Arizona State University (Publisher)
Created2020