Browsing by Subject "RNAi"
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Item Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses(2016-07-21) Burke, James Michael; Sullivan, Christopher S.; Dudley, Jaquelin; Upton, Jason; Ehrlich, Lauren; Russell, Rick; Ulug, EminSome eukaryotic viruses express small RNAs called microRNAs (miRNAs) to regulate host and viral gene expression. Due to their small genomic footprint, ability to regulate numerous genes, and lack of immunogenicity, miRNAs are an apt gene regulatory mechanism for viruses. Nevertheless, few viral miRNAs have been studied in vivo and the biological functions of most remain unknown. All known viral miRNAs are generated via host machinery. Most viral miRNAs mature through the canonical miRNA biogenesis pathway, whereas a few viruses generate miRNAs using noncanonical miRNA mechanisms. The noncanonical biogenesis mechanisms and the variety of miRNA alleles associated with some viruses provide powerful tools for probing mammalian small RNA biology. In this dissertation, I analyze the biogenesis and function of miRNAs encoded by diverse tumor viruses. In chapter 2, I utilize the Simian virus 40 (SV40) primary miRNA (pri-miRNA) as a system to provide new mechanistic insights relevant to canonical pri-miRNA processing. This work reveals that multiple pri-miRNA structures coordinate processing by the Microprocessor complex. In chapter 3, I characterize the biogenesis of noncanonical miRNAs encoded by bovine leukemia virus (BLV), a deltaretrovirus. This work demonstrates that the BLV pre-miRNAs are directly transcribed by RNA polymerase III from proviral genomes, circumventing the requirement of mRNA cleavage for miRNA production. In chapter 4, using BLV and Adenoviral miRNAs, I demonstrate that dual-specificity phosphatase 11 (DUSP11) promotes accumulation and activity of small RNAs derived from diverse 5´-triphosphorylated precursors in the RNA-induced silencing complex (RISC). In chapter 5, I develop a novel method to express short hairpin RNAs (shRNAs) using the architecture of the BLV miRNA genes, thereby decreasing the template space required for shRNA expression. This work applies to gene silencing strategies where smaller gene cassettes are desirable. In chapter 6, I report that the miRNAs encoded by murine polyomavirus (MuPyV) are not required for viral persistence. Instead, the MuPyV miRNAs promote viral shedding during the acute phase of infection in vivo. This work provides a fundamental understanding of the functional role of polyomavirus-encoded miRNAs. Combined, this work advances the fields of experimental gene silencing, small RNA biology, and virus-host interactions.Item The discovery of novel proteins involved in cell polarity and the PAR complex in c. elegans(2021) Koh, Alexander; Dickinson, DanielCell polarity is a phenomenon that occurs within nearly all organisms. Characterized by the localization of different proteins to different ends of a cell, this process is extremely important for proper biological development. Although studying this mutually antagonistic binding is a growing field of study, cell polarity as a process is still immensely complex and how exactly it is established is still not well known. However, by focusing on known proteins involved in the polarity process, mainly the PAR protein system, other proteins that interact with the PAR complex can be studied for individual effects on cell polarity. The PAR complex was first discovered in c. elegans Multiple experiments were conducted on multiple polarity proteins to identify new potential binding partners to the proteins in the PAR complex. These experiments stem from the cell polarity proteins in the PAR complex, which include aPKC, PAR-6, and PAR-3. A list of proteins from that interact with aPKC was compiled using mass spectrometry pull down. These proteins were studied using RNAi to knock out the gene in C. elegans, and the effects on cell polarity in developing embryos were observed. These genes were then genetically engineered to be injected into worms using Gibson assembly and CRISPR-Cas9 to look for further effects. Furthermore, tests were conducted on strains involving PAR-6. Results show that perhaps the nematode database does not account for an extra intron portray protein sequence, which could lead to further development on the functions of PAR-6, and thus, cell polarity. Finally, mass spectrometry pull down was done on PAR-3 monomer strains due to previous research showing potential of these PAR-3 monomers in having an essential function in C. elegans. This resulted in a list of pulled-down novel proteins, ultimately in which some could potentially affect polarity in new and unknown ways.Item Identifying functions of Down syndrome-related genes using RNA interference in C. elegans(2010-12) Griffith, Allison Mooney; Pierce-Shimomura, Jonathan T.; Marcotte, Edward M.Down syndrome is one of the most common genetic disorders, resulting in a range of neurological and neuromuscular disabilities. Although the presence of specific disabilities varies among individuals with Down syndrome, all individuals with Down syndrome are born with hypotonia (low muscle tone) and over half with congenital heart defects. Later in life, all individuals demonstrate intellectual disabilities to varying degrees, while many also develop early-onset Alzheimer’s disease. While the cause of Down syndrome is known to be a triplication of the 21st chromosome, it is unknown how this extraneous genetic material causes the development of these phenotypes. We have begun research into the biological basis of these disabilities using the tiny nematode, Caenorhabditis elegans as a genetic model. We used the technique RNA interference (RNAi), which allows us to study the in vivo function of genes by knocking down their expression one at a time in a living, behaving animal. We have used this technique to systematically study the in vivo function for genes involved in Down syndrome. To this end, we identified and knocked down C. elegans genes with sequence similarity to 67% of genes on the human 21st chromosome genes. We used these RNAi-treated worms to investigate the neuromuscular function of human chromosome 21 gene equivalents by assaying locomotion and pharyngeal pumping in a blinded screen. We used locomotion as a measure of neurological and neuromuscular function, while we used pharyngeal pumping as a model for cardiac function. We also performed an aldicarb screen to examine the role of some of these genes in the function of the synapse. Our experiments have provided valuable insight into the in vivo function of the vast majority of genes on the human 21st chromosome. This will be vital to identify genes that are potentially involved in eliciting Down syndrome-related phenotypes, laying the groundwork for further studies into the neurobiology of Down Syndrome.Item The role of a viral microRNA and RNA interference during viral replication in mammalian cells(2012-12) Seo, Gil Ju; Sullivan, Christopher S.RNA interference (RNAi) is an evolutionarily conserved process that regulates gene expression. Host cells and viruses interact in many ways, including through miRNAs and RNAi. Viral miRNAs are encoded when viruses, specially including the the polyoma and herpes families, are transcribed in the nucleus. Some viral miRNAs function to regulate host or viral gene expression. Most viral miRNAs’ functions are not known, however, in great detail. A miRNA can be encoded late during infection, as it is by SV40, a model polyomavirus. This downregulates early viral gene expression by directing mRNA RISC-mediated cleavage. As more polyomaviruses are discovered that are associated with human disease, it becomes more important to understand their function and to uncover whether these emerging viruses encode miRNAs. The work presented here shows the discovery of several viral miRNAs in human polyomaviruses—JCV, BKV, and MCV. In addition, I found that viral miRNAs have the evolutionarily conserved function of negatively regulating viral early gene transcripts at a late stage in the infection. During viral replication, viruses utilize the miRNA components of RNAi. However, in invertebrate organisms RNAi also actively defends against viral infection. It is still being debated, though, whether RNAi plays an antiviral role in mammalian cells. Should it be true that RNAi is an antiviral response in mammalian cells, then what is predicted by such a scenario is inconsistent with my studies. I have found that RNAi is strongly inhibited in the early stages after viral infection. Studies with a chemical mimic of viral infection (poly I:C) imply that the innate cellular immune response is responsible for this inhibition. I investigated the molecular changes, in response to viral infection, (e.g. poly ADP-ribosylation of Ago2) in the RNA-induced silencing complex (RISC). I determined that the inhibition of RNAi is brought about by components of the innate response. Completion of this study details a previously unknown “cross talk” between RNAi and the host innate immune response in mammalian cells. Furthermore, I found mir-17 family attenuates a subclass of interferon-stimulated genes. An understanding of viral miRNA and RNAi offers a clue as to we can use molecular intervention for viral infections.