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Description
This project aims to tackle two perspectives: to design and express an enzyme that can perform single-molecule modifications for identification, and to determine the inclusion of the last adenosine in mature mRNAs within the metazoan, Caenorhabditis elegans. Starting with the first perspective, the enzymatic group that was utilized was methyltransferases. Methyltransferases have gained great interest in biotechnology and academia due to their ability to make single-molecule modifications to a wide variety of biomolecules, ranging from proteins to RNA. Of these methyltransferases, the subset that has the greatest interest for this study are RNA methyltransferases. Of the known RNA methyltransferases, human METTL16 was chosen for this project, due to its ability to modify adenosines at the N6 position (m6A), specificity for its consensus motif, and its promise in chimeric enzymatic complexes. As a result of these properties, this study looks to design METTl16-based complexes for the purpose of identifying single nucleotides in RNA.
The second perspective involves pre-mRNA cleavage and polyadenylation of the 3’ untranslated region (3’UTR). Cleavage of pre-mRNAs within C.elegans appears to prefer an adenosine, leading to the term “terminal adenosine” (terminal-A). Since RNA cleavage and polyadenylation is highly conserved across metazoans, we can utilize the model system, C. elegans, to apply our findings to humans. Utilizing METTL16’s ability to modify adenosines, it is theorized that it may be possible to modify the terminal-A in vivo within C. elegans. To confirm the functionality and utilization of METTL16, a novel methodology is currently being developed called the terminal adenosine methylation (TAM) assay. The TAM assay takes advantage of METTL16’s N-terminal RNA binding domain (RBD) and methyltransferase domain – called the “core” – to methylate the terminal adenosine of probe mRNA transcripts prior to cleavage in vivo. To determine if the adenosine is present within mature mRNAs, sequencing will determine if there is a m6A present, confirming that CPSF-3 cleaves either upstream or downstream of the terminal-A.
Ultimately, this project focuses on designing METTL16 complexes for mRNA modification, testing the functionality of these constructs in vitro, and developing transgenic C. elegans strains to express the METTL16 complexes. The bioconjugation capabilities of RNA methyltransferases allow for concepts such as the TAM assay to be viable, as well as make way for future prospects of methyltransferases as a biotechnical tool.
ContributorsMurray, Jillian (Author) / Mangone, Marco (Thesis director) / Lapinaite, Audrone (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor)
Created2024-05