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Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate research on the properties of yeast Gle1 (yGle1) with those of human Gle1 (hGle1). Research at Vanderbilt University in 2016,

Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate research on the properties of yeast Gle1 (yGle1) with those of human Gle1 (hGle1). Research at Vanderbilt University in 2016, which provides the research basis of this thesis, suggests that the coiled-coil domain of yGle1 is best crystallized in dicationic aqueous conditions of pH ~8.0 and 10\u201420% PEG 8000. Further exploration of crystallizable microconditions revealed a favorability toward lower pH and lower PEG concentration. Following the discovery of the protein's native crystallography conditions, a comprehensive meta-analysis of scientific literature on Gle1 was conducted on the association of Gle1 mutations with neuron disease.
ContributorsGaetano, Philip Pasquale (Author) / Foy, Joseph (Thesis director) / Dawson, T. Renee (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description

Molecular engineering is an emerging field that aims to create functional devices for modular purposes, particularly bottom-up design of nano-assemblies using mechanical and chemical methods to perform complex tasks. In this study, we present a novel method for constructing an RNA clamp using circularized RNA and a broccoli aptamer for

Molecular engineering is an emerging field that aims to create functional devices for modular purposes, particularly bottom-up design of nano-assemblies using mechanical and chemical methods to perform complex tasks. In this study, we present a novel method for constructing an RNA clamp using circularized RNA and a broccoli aptamer for fluorescence sensing. By designing a circular RNA with the broccoli aptamer and a complementary DNA strand, we created a molecular clamp that can stabilize the aptamer. The broccoli aptamer displays enhanced fluorescence when bound to its ligand, DFHBI-1T. Upon induction with this small molecule, the clamp can exhibit or destroy fluorescence. We demonstrated that we could control the fluorescence of the RNA clamp by introducing different complementary DNA strands, which regulate the level of fluorescence. Additionally, we designed allosteric control by introducing new DNA strands, making the system reversible. We explored the use of mechanical tension to regulate RNA function by attaching a spring-like activity through the RNA clamp to two points on the RNA surface. By adjusting the stiffness of the spring, we could control the tension between the two points and induce reversible conformational changes, effectively turning RNA function on and off. Our approach offers a simple and versatile method for creating RNA clamps with various applications, including RNA detection, regulation, and future nanodevice design. Our findings highlight the crucial role of mechanical forces in regulating RNA function, paving the way for developing new strategies for RNA manipulation, and potentially advancing molecular engineering. Although the current work is ongoing, we provide current progress of both theoretical and experimental calculations based on our findings.

ContributorsJoseph, Joel (Author) / Yan, Hao (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Lapinaite, Audrone (Committee member) / Barrett, The Honors College (Contributor) / Historical, Philosophical & Religious Studies, Sch (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
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 a large complex known as the Cleavage and Polyadenylation Complex (CPC). Its central subcomplex, the Cleavage and Polyadenylation Specificity Factor

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.
ContributorsGallante, Christina (Author) / Mangone, Marco (Thesis director) / Sharma, Shalini (Committee member) / Hrach, Heather (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05