Matching Items (6)

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High-throughput identification of miRNA targets in the 3`UTRs of the soil nematode C. elegans

Description

miRNAs are short non-coding regulatory RNAs that have an important roles in a wide range of biological processes. Dysfunction of miRNA regulation has also been shown to occur in diseases

miRNAs are short non-coding regulatory RNAs that have an important roles in a wide range of biological processes. Dysfunction of miRNA regulation has also been shown to occur in diseases such as cancer. Despite the widespread influence of miRNAs in these contexts, the vast majority of miRNA targets are poorly characterized. The aim of this research project was to gain a better understating of miRNA targeting by using the model organism C. elegans. In order to do this I adapted a novel high-throughput assay to detect miRNA targets for use with the C. elegans 3`UTRome. As a proof of principle I performed this assay on 96 C. elegans 3`UTRs using high-throughput techniques. The results revealed miRNA interactions with two predicted 3`UTR targets for the miRNA lin-4 and ten unpredicted targets. The results also corroborated previous findings that certain worm miRNAs require special modifications to be expressed in human cells.

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Created

Date Created
  • 2013-12

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C. elegans as a Model to Study Muscle Specific Changes in Duchenne Muscle Dystrophy

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

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.

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Date Created
  • 2016-05

Characterization of mRNA Transcription Termination and Cleavage in C. elegans

Description

In eukaryotes, most messenger RNA precursors (pre-mRNA) undergo extensive processing, leading to the cleavage of the transcript followed by the addition of a poly(A) tail. This process is executed by

In eukaryotes, most messenger RNA precursors (pre-mRNA) undergo extensive processing, leading to the cleavage of the transcript followed by the addition of a poly(A) tail. This process is executed by a large complex known as the Cleavage and Polyadenylation Complex (CPC). Its central subcomplex, the Cleavage and Polyadenylation Specificity Factor (CPSF) complex is responsible for recognizing a short hexameric element AAUAAA located at the 3’end in the nascent mRNA molecule and catalyzing the pre-mRNA cleavage. In the round nematode C. elegans, the cleavage reaction is executed by a subunit of this complex named CPSF3, a highly conserved RNA endonuclease. While the crystal structure of its human ortholog CPSF73 has been recently identified, we still do not understand the molecular mechanisms and sequence specificity used by this protein to induce cleavage, which in turn would help to understand how this process is executed in detail. Additionally, we do not understand in additional factors are needed for this process. In order to address these issues, we performed a comparative analysis of the CPSF3 protein in higher eukaryotes to identify conserved functional domains. The overall percent identities for members of the CPSF complex range from 33.68% to 56.49%, suggesting that the human and C. elegans orthologs retain a high level of conservation. CPSF73 is the protein with the overall highest percent identity of the CPSF complex, with its active site-containing domain possessing 74.60% identity with CPSF3. Additionally, we gathered and expressed using a bacterial expression system CPSF3 and a mutant, which is unable to perform the cleavage reaction, and developed an in vitro cleavage assay to test whether CPSF3 activity is necessary and sufficient to induce nascent mRNA cleavage. This project establishes tools to better understand how CPSF3 functions within the CPC and sheds light on the biology surrounding the transcription process as a whole.

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Created

Date Created
  • 2020-05

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Study of the Requirements of RNA Cleavage and Polyadenylation in C. elegans

Description

Cleavage and polyadenylation is a step in mRNA processing in which the 3’UTR is cleaved and a polyA tail is added to create a final mature transcript. This process relies

Cleavage and polyadenylation is a step in mRNA processing in which the 3’UTR is cleaved and a polyA tail is added to create a final mature transcript. This process relies on RNA sequence elements that guide a large multimeric protein complex named the Cleavage and Polyadenylation Complex to dock on the 3’UTR and execute the cleavage reaction. Interactions of the complex with the RNA and specific dynamics of complex recruitment and formation still remain largely uncharacterized. In our lab we have identified an Adenosine residue as the nucleotide most often present at the cleavage site, although it is unclear whether this specific element is a required instructor of cleavage and polyadenylation. To address whether the Adenosine residue is necessary and sufficient for the cleavage and polyadenylation reaction, we mutated this nucleotide at the cleavage site in three C. elegans protein coding genes, forcing the expression of these wt and mutant 3’UTRs, and studied how the cleavage and polyadenylation machinery process these genes in vivo. We found that interrupting the wt sequence elements found at the cleavage site interferes with the cleavage and polyadenylation reaction, suggesting that the sequence close to the end of the transcript plays a role in modulating the site of the RNA cleavage. This activity is also gene-specific. Genes such as ges-1 showed little disruption in the cleavage of the transcript, with similar location occurring in both the wt and mutant 3’UTRs. On the other hand, mutation of the cleavage site in genes such as Y106G6H.9 caused the activation of new cryptic cleavage sites within the transcript. Taken together, my experiments suggest that the sequence elements at the cleavage site somehow participate in the reaction to guide the cleavage reaction to occur at an exact site. This work will help to better understand the mechanisms of transcription termination in vivo and will push forward research aimed to study post-transcriptional gene regulation in eukaryotes.

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Created

Date Created
  • 2019-05

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Mechanisms of miRNA-based gene regulation in C. elegans and human cells

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

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.

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Agent

Created

Date Created
  • 2019

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Degeneration in miniature: history of cell death and aging research in the twentieth century

Description

Once perceived as an unimportant occurrence in living organisms, cell degeneration was reconfigured as an important biological phenomenon in development, aging, health, and diseases in the twentieth century. This dissertation

Once perceived as an unimportant occurrence in living organisms, cell degeneration was reconfigured as an important biological phenomenon in development, aging, health, and diseases in the twentieth century. This dissertation tells a twentieth-century history of scientific investigations on cell degeneration, including cell death and aging. By describing four central developments in cell degeneration research with the four major chapters, I trace the emergence of the degenerating cell as a scientific object, describe the generations of a variety of concepts, interpretations and usages associated with cell death and aging, and analyze the transforming influences of the rising cell degeneration research. Particularly, the four chapters show how the changing scientific practices about cellular life in embryology, cell culture, aging research, and molecular biology of Caenorhabditis elegans shaped the interpretations about cell degeneration in the twentieth-century as life-shaping, limit-setting, complex, yet regulated. These events created and consolidated important concepts in life sciences such as programmed cell death, the Hayflick limit, apoptosis, and death genes. These cases also transformed the material and epistemic practices about the end of cellular life subsequently and led to the formations of new research communities. The four cases together show the ways cell degeneration became a shared subject between molecular cell biology, developmental biology, gerontology, oncology, and pathology of degenerative diseases. These practices and perspectives created a special kind of interconnectivity between different fields and led to a level of interdisciplinarity within cell degeneration research by the early 1990s.

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Created

Date Created
  • 2013