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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.
ContributorsSteber, Hannah Suzanne (Author) / Mangone, Marco (Thesis director) / Harris, Robin (Committee member) / LaBaer, Joshua (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Tissue regeneration is a complex process that activates both developmental and metabolic signaling pathways (Kashio & Miura, 2020). The wing imaginal disc in Drosophila melanogaster has been invaluable in discerning what pathways are activated during tissue regeneration, which is typically done by genetically or physically wounding the wing disc and using fluorescent markers to track different signals. However, despite its importance in other regeneration contexts (Tafesh-Edwards & Eleftherianos, 2020), immune signaling has not been well studied in this tissue. Furthermore, what we do know about tissue regeneration and immune signaling is specific to apoptotic cellular death, less is known about other types of cellular death, such as necrotic cellular death and the consequent signaling systems that result from necrosis. Drosophila have an open immune system and only possess innate immunity (Pastor-Pareja et al., 2008), making them an ideal model to study hemocyte involvement in tissue regeneration. Hemocytes are equivalent to blood cells in vertebrates, and are involved in immunological response (Kurucz et al., 2003). In this work, we observed hemocyte accumulation during injury-induced regeneration. Cellular damage was induced using a genetic ablation system known as DUAL Control, with hemipterous CA and GluR1 used to induce apoptotic and necrotic cell death respectfully. We have discovered that while hemocytes are recruited to the wing disc upon both apoptotic and necrotic injury, necrotic tissue has more hemocytes adhered than apoptotic tissue. The difference in adherence could be due to basement membrane integrity being damaged more severely in necrotic discs than apoptotic discs. Our results show that hemocytes are attracted to wing discs that have undergone necrotic damage, indicating that the immune system plays some sort of role in necrotic cellular death. Though the immune response to different types of tissue damage in Drosophila is much simpler than in vertebrate models, there are many similarities between the two, and could lead to research involving human immune signaling as it pertains to regeneration.
ContributorsZustra, Ayla (Author) / Harris, Robin (Thesis director) / Gile, Gillian (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05
Description
Tuberous sclerosis complex (TSC) is a rare genetic disease caused by heterozygous dominant mutations in the TSC1 or TSC2 genes that affects 1/6000 newborns (Curatalo et al., 2002; de Vries & Howe, 2007). TSC has a variety of clinical manifestations ranging from hypomelanotic macules to neurological conditions such as epilepsy (Neuman & Henske, 2011; de Vries & Howe, 2007). In cases where the TSC mutations are inherited from parent to offspring (familial TSC)- the child can still exhibit more severe symptoms despite having the same TSC mutation as the parent, a phenomenon known as intrafamilial phenotypic variability (IPV) (Curatalo et al, 2002). We hypothesize that the variants in genes of the mTOR signaling pathway (genetic modifiers) may enhance or suppress mTOR pathway activity, resulting in IPV. Patient derived primary fibroblasts cell lines from two families exhibiting IPV were studied as well as an unrelated control cell line. We identified variants in IRS1, FZD5, and PIK32CG genes from children with severe phenotype in one family and variants in PIK3R3, TNFRSF19, and EIF4G1 in a severe child in another pathway. We explored the functional impact of these genes on mTOR pathway activity.
ContributorsFry, Hannah (Author) / Rangasamy, Sampath (Thesis director) / Harris, Robin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Watts College of Public Service & Community Solut (Contributor)
Created2022-05