STRUCTURAL AND FUNCTIONAL STUDIES OF H. VOLCANII BOX C/D PROTEINS AND ROLES FOR HUMAN PUS10 BEYOND PSEUDORURIDINE SYNTHESIS
Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
RNA in all forms of life contain a myriad of post-transcriptional modifications. These modifications are important for processing and structural reasons, and includes 2’-O-methylation and pseudouridylation. Some of these modifications are the product of stand-alone proteins and others are the product of RNA-protein complexes.In eukaryotic and archaeal cells, the Box C/D ribonucleoprotein complex is one of the complexes responsible for 2’-O-methylation activity. In Archaea, this complex consists of the enzymatic protein, Fibrillarin, plus two other structural proteins, Nop5 and L7Ae, along with a guide RNA, which all come together to modify a specific target RNA sequence. A methyl group is added to the 2’ hydroxyl of the nucleotide’s ribose sugar. These modifications are found both in ribosomal RNA and tRNAs. This work focuses on the interactions between Nop5, Fibrillarin, and the guide RNA used in this complex. The objective was to identify important amino acid sequences in these proteins which are essential for the operation of the complex. The size of the archaeal Box C/D complex is also investigated using size chromatography.Similarly, Pus10 is one protein responsible for pseudouridylation in Archaea and eukaryotes. Pseudouridylation is the isomerization of uridine (U) to pseudouridine (Ψ). Pus10 is known to produce Ψ54 and Ψ55 in the TΨC loop of some archaeal and eukaryotic tRNAs. However, current research suggests it could have additional jobs in the cellular lifecycle, such as roles in apoptosis and the regulation of eukaryotic cell cycle. During mammalian cell apoptosis, Pus10 translocates from the nucleus to the cytoplasm and is believed to be involved in cytochrome c release. This is suspected to be related to Caspase-3’s role in apoptosis. Caspase-3 is activated in both the extrinsic and intrinsic apoptotic pathways. It has been shown that activation of the extrinsic apoptotic pathway via TRAIL affects the localization of Pus10. Here we test Pus10’s actions in cells under the effect of intrinsic apoptotic pathway compounds.Pus10 has also shown to have an effect on cell proliferation. Cells in which Pus10 has been depleted show increased growth rates. Here we investigate expression levels of proteins involved in cell cycle regulation, in both wild-type and Pus10-depleted cells, and attempt to compile a model of how Pus10 could be interacting with this system, using RNA-Seq, qPCR, and ChIP.SARS-CoV-2, also known as COVID-19, is a coronavirus that quickly became a pandemic in late 2019, early 2020. In the two years since then, the virus has undergone many mutations. By tracking the spread of these mutations at a national or regional level, by studying the spread pattern, we can make predictions, and possibly even prevent the next pandemic.
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