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Browsing by Author "Wilusz, Carol J., committee member"

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    Analysis of virus-derived small RNAs reveals that the RNA silencing response to flavivirus infection differs dramatically between C6/36 and Aag2 mosquito cell lines
    (Colorado State University. Libraries, 2010) Scott, Jaclyn Christine, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Wilusz, Carol J., committee member; Peersen, Olve, committee member
    The exogenous small RNA pathway has been shown to be an important antiviral defense in mosquitoes against arboviruses such as dengue virus (DENV), but little is known about how the pathway and the virus interact in the cell. The studies described in this dissertation examine the how small RNA pathways interact with DENV and a mosquito-only flavivirus, cell-fusing agent virus (CFAV), in mosquito cell cultures. Deep sequencing of virus-specific small RNAs in Aedes aegypti Aag2 cells indicates that DENV2 is targeted by the exogenous RNA interference (RNAi) pathway in this cell line, which is consistent with the DENV2-specific small RNAs seen in DENV2-infected A. aegypti mosquitoes. When the DENV2-specific small RNAs from the Aedes albopictus C6/36 cell line were analyzed, the size and polarity of the small RNAs was not consistent with the exogenous small interfering RNA (siRNA) pathway. Further molecular analysis of the C6/36 cell line indicated that it appears to lack functional Dicer2 processing of long double-stranded RNA (dsRNA). CFAV small RNAs were also discovered in the Aag2 cell line during the deep sequencing analysis. It appears that this cell line is persistently infected with this mosquito-only flavivirus, and the virus is also targeted by the exogenous siRNA pathway in the cells. Sequence comparisons between CFAV and DENV2 RNA did not show long regions of sequence identity between the two viruses, indicating that a sequence-specific mechanism for virus-derived small RNAs from one virus to interfere with replication of the other virus during dual infections seems unlikely. The C6/36 cell line was inadvertently infected with CFAV, but the CFAV-specific small RNAs in C6/36 cells did not appear to be generated from the exogenous siRNA pathway, consistent with the DENV2-specific small RNAs in this cell line. The larger sized, mostly positive sense virus-specific small RNAs found in the C6/36 cells suggest that virus infections may be targeted by another small RNA pathway (such as the piwi-interacting pathway) in this cell line. These studies provide a better understanding of the interactions of DENV2 with the mosquito antiviral RNAi pathway in infected mosquito cells and have revealed a dysfunctional RNAi pathway in the C6/36 cell line. This work also provides a basis for further studies examining the interactions between mosquito-only flaviviruses, arboviruses and the antiviral RNAi pathway.
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    Commandeering of the cellular HuR protein by alphaviruses affects the regulation of host post-transcriptional gene expression
    (Colorado State University. Libraries, 2013) Barnhart, Michael D., author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., committee member; Laybourn, Paul J., committee member
    It was previously shown that cellular HuR protein binds to a U-rich region in the 3'UTR of Sindbis virus RNA resulting in stabilization of viral transcripts and increased replication efficiency. While the presence of this U-rich region is generally conserved among alphaviruses, a subset lacks a typical U-rich region. The 3'UTR of two alphaviruses - Ross River virus and Chikungunya virus - that do not contain a typical U-rich region were tested for HuR interactions by Electrophoretic Mobility Shift Assay. HuR protein bound these 3'UTRs with nanomolar affinities, similar to what was observed for the U-rich region of Sindbis virus. These observations demonstrate that the critical role for HuR-mediated viral RNA stabilization is likely a conserved property of most, if not all, members of the virus family. By analyzing deletion derivatives, we mapped the novel HuR binding sites in these two viruses to specific regions in their 3'UTR. Next, we uncovered four novel aspects of virus-host interaction and pathogenesis related to the high affinity interaction between the 3'UTR of alphaviruses and the cellular HuR protein. First, HuR protein, which is usually localized predominantly to the nucleus, dramatically accumulates in the cytoplasm during Sindbis virus (SinV) infection. Studies involving the transfection of constructs that express viral 3'UTR RNA fragments indicated that the mechanism of induction of HuR accumulation to the cytoplasm in infected cells is due to the viral RNA acting as a sponge for the protein. Second, HuR interaction with numerous cellular mRNAs was found to be drastically decreased during a SinV infection and was associated with dramatic destabilization of the cellular transcripts as determined by mRNA half-life analysis. Third, we found that the reduced amounts of free HuR during a SinV infection results in the increased targeting of mRNAs by miRNAs. Together, these data indicate that in the process of commandeering the cellular HuR protein for its own use, alphaviruses are also effectively destabilizing numerous cellular mRNAs. Interestingly, many of the cellular mRNAs affected by alphaviruses play key roles in inflammation, innate immune responses and other fundamental cellular processes. Finally, we observed a novel effect of SinV infection on alternative polyadenylation of cellular transcripts. This is likely a direct result of sequestration of the HuR protein in the cytoplasm by the virus, preventing the protein from influencing nuclear polyadenylation site choice. Intriguingly, SinV infection influences the poly(A) site choice of the HuR pre-mRNA, favoring a more translatable isoform to promote the overexpression of this viral host factor. Therefore, the alphaviral-induced alterations in cellular mRNA stability and polyadenylation identified in this thesis may play a very important but underappreciated role in pathogenesis.
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    In vivo regulation of chromatin dynamics by Saccharomyces cerevisiae histone chaperone Nap1
    (Colorado State University. Libraries, 2011) Barker, Kristi Leigh, author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Wilusz, Carol J., committee member
    Eukaryotic cells must organize massive amounts of DNA into the nucleus. In order to accomplish this, the DNA must be compacted into a highly ordered structure known as chromatin. The basic, repeating unit of chromatin is the nucleosome, which consists of two copies of each histone (H2A, H2B, H3 and H4) and organizes 147 base pairs of DNA. Due to its highly compact nature, nucleosomes must be removed during gene expression in order for the transcription machinery to access the DNA. Shuttling of nucleosomes on and off DNA is mediated by a group of proteins known as histone chaperones. Importantly, histone chaperones interact with another family of chromatin remodeling complexes known as histone acetyltransferases (HATs). Acetylation of histones is correlated with the active transcription of genes. The work presented here explores the dynamics and kinetics of histone H3 occupancy and acetylation of histone H3-K9 and H3-K14 in a wild-type strain and strains deleted for three known histone chaperones (Nap1, Vps75 and Asf1) of the yeast Saccharomyces cerevisiae at the well characterized galactose inducible genes. This data offers insight into the epigenetic regulation of chromatin as well as possible mechanisms for the histone chaperones surveyed.
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    Oxidative and energetic stress: regulation of Nrf2 and mitochondrial biogenesis for slowed aging interventions
    (Colorado State University. Libraries, 2013) Bruns, Danielle Reuland, author; Hamilton, Karyn L., advisor; Miller, Benjamin F., advisor; Wilusz, Carol J., committee member; Pagliassotti, Michael J., committee member
    The following dissertation describes a series of experiments with the overall aim to understand the cellular energetic and oxidative stresses associated with aging, and to investigate treatments which may attenuate these stresses and promote healthspan. The specific aims of the four sets of experiments were 1) to determine if treatment with the phytochemicals in Protandim activates Nrf2 and 2) the mechanisms by which this activation occurs; 3) to assess sexually dimorphic Nrf2 signaling across three rodent models of longevity; and 4) to determine if mitochondrial related proteins are preferentially translated under energetic stress. In Experiments #1 and #2, we found that phytochemicals activate Nrf2 and protect cells against oxidant stress. None of the mechanisms we investigated appear to be responsible for phytochemical-induced Nrf2 activation, and continued investigations must be undertaken to identify how Protandim robustly induces Nrf2 nuclear accumulation. In Experiment #3, we found that Nrf2 signaling was not consistently upregulated in tissues from long-lived models compared to controls, but we did elucidate important sex differences, with female mice generally displaying greater Nrf2 signaling than male mice. We believe this finding, in the context of sexual dimorphism in aging, warrants future investigations into Nrf2, stress resistance, and longevity between males and females. In Experiment #4, we found that mitochondrial proteins were preferentially translated upon pharmaceutical energetic stress, and that this selective translation occurred in the vicinity of the mitochondria. Our results indicate activation of Nrf2 protects cells against oxidant stress, and may be a therapeutic target for cardiovascular diseases and other age-related diseases. Further, we assess selective translation of mitochondrial proteins during energetic stress as a means of understanding how energetic stress mimetics selectively facilitate the translation of key mitochondrial proteins. Taken together, these studies provide the basis for future work aimed at attenuating diseases with oxidant stress and mitochondrial dysfunction components.
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    Protein synthesis in slowed aging: insights into shared characteristics of long-lived mouse models
    (Colorado State University. Libraries, 2014) Drake, Joshua Chadwick, author; Miller, Benjamin F., advisor; Hamilton, Karyn L., advisor; Wilusz, Carol J., committee member; Hickey, Matthew S., committee member
    The following dissertation describes a series of experiments with the overall aim to understand the role that changes in protein synthesis have in slowed aging. The specific aims of the three sets of experiments were 1) to determine if chronic administration of the mTORC1 inhibitor rapamycin to mice increases proteostatic mechanisms in skeletal muscle, heart, and liver; 2) to determine if an underdeveloped anterior pituitary, caused by deletion of the Pit-1 gene in mice, increases proteostatic mechanisms in skeletal muscle, heart, and liver of long-lived Snell dwarf mice; 3) and to determine if transient nutrient restriction during the suckling period in mice (i.e. crowded litter), increases proteostatic mechanisms in skeletal muscle, heart, and liver later in life. In Experiment #1 we found that mitochondrial proteins were preferentially synthesized in skeletal muscle and that global protein synthesis in the heart was maintained despite reduced cellular proliferation and mTORC1 activity in mice fed rapamycin compared to normal diet controls. Originally we determined that these data were indicative of an improved somatic maintenance of skeletal muscle mitochondria and the heart proteome. Since we could not account for changes to other energetic processes (e.g. metabolism), we reasoned that our data was more consistent with proteostasis, a component of somatic maintenance. In Experiment #2 we developed a novel method for assessing proteostasis and determined that Snell dwarf mice had an increase in proteostatic mechanisms across sub-cellular fractions within skeletal muscle and heart compared control mice, despite differential rates of protein synthesis in the face of decreased mTORC1. Together with our previous investigations into rapamycin fed and caloric restriction models of long-life we concluded that increased proteostatic mechanisms may be a shared characteristic of models of slowed aging. In Experiment #3 we demonstrate that the crowded litter mouse transitions from growth to maintenance as it ages. Furthermore, in the crowded litter mouse, we demonstrate that proteostasis is not dependent upon decreased mTORC1. Our results indicate that decreased mTORC1 does not necessarily correlate to decreases in protein synthesis across all sub-cellular fractions. Discerning which proteins and the mechanism(s) of how specific proteins can be preferentially synthesized despite decreases in protein synthesis in other fractions and decreased mTORC1, may give further insight into characteristics of slowed aging. Further, we demonstrate that increases in proteostasic mechanisms are a shared characteristic of multiple unique models of slowed aging and therefore, provides a basis for future work aimed at slowing the aging process.
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    Role of HuR, AUF1 and zeta-crystallin in mediating pH-responsive increase in renal phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in kidney cells
    (Colorado State University. Libraries, 2012) Gummadi, Lakshmi, author; Curthoys, Norman P., advisor; Nyborg, Jennifer K., committee member; Laybourn, Paul J., committee member; Wilusz, Carol J., committee member
    The maintenance of blood acid-base balance is essential for survival. However, metabolic acidosis is a common clinical condition that is characterized by a significant decrease in plasma pH and bicarbonate concentration. This alteration is caused by genetic or acquired defects in metabolism, in renal handling of bicarbonate, and in the excretion of titratable acid. In addition, metabolic acidosis could pose a secondary complication in patients with cachexia, trauma, uremia, end stage renal disease, osteomalacia, HIV infection and in patients with degenerative diseases. Increased renal ammoniagenesis and gluconeogenesis from plasma glutamine constitute an essential physiological response to metabolic acidosis that partially restores acid-base balance. A portion of this adaptive response is the rapid and pronounced increase in the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) that occurs within the renal proximal convoluted tubule. Previous in vitro biochemical studies have mapped the binding of AUF1, HuR and ζ-crystallin (ζ-Cryst) to various AU-rich sequences within the 3'UTR of PEPCK mRNA. This response is reproduced in LLC-PK1-F+9C cells that are treated with acidic (pH 6.9) medium. It is mediated, in part, by stabilization of PEPCK mRNA. Here I have used a combination of approaches to characterize the dynamic interaction of trans-acting factors with the cis-acting elements in mediating the pH-responsive stabilization of PEPCK mRNA. In chapter III I show that binding of HuR and AUF1 have opposite effects on basal expression, but their co-ordinate interaction is required to mediate the pH-responsive adaptation. Consistent with this, while the individual recruitment of a chimeric protein containing the MS2 coat protein and either HuR or p40AUF1 failed to produce a pH-responsive stabilization, the concurrent expression of both chimeric proteins was sufficient to produce a pH-responsive increase in the half-life of the reporter mRNA. This study also demonstrated that HuR and AUF1 underwent profound altered post-translational modifications when LLC-PK1-F+9C cells were switched from basal to acid-pH medium conditions. In Chapter IV I went on to demonstrate that HuR makes direct interaction with PEPCK mRNA and that HuR/ AUF1 form hetero-oligomeric complex in an RNA-dependent manner. Finally in Chapter V I investigated the functional significance of the ζ-Cryst binding to the PEPCK-3'UTR. These experiments suggested that ζ-Cryst may serve as a key co-factor along with HuR and AUF1 to restrict the basal expression and that only HuR and AUF1 are required for the pH-responsive increase of PEPCK mRNA. Based upon the findings of the current study, I proposed a model depicting the co-ordinate role of HuR, AUF1 and ζ-Cryst in post-transcriptional regulation of PEPCK mRNA turnover and, more importantly in mediating the sustained pH-responsive increase of PEPCK mRNA. Under normal acid-base conditions, phosphorylated HuR, covalently modified AUF1 and ζ-crystallin are co-recruited to the 3'-UTR of PEPCK mRNA and may form a complex through direct protein-protein interactions. The binding of the three RNA-binding proteins leads to recruitment of a deadenylase that removes the poly-A tail and leads to the subsequent decapping and 5'→3' decay of the deadenylated PEPCK mRNA. However onset of metabolic acidosis leads to alterations in post-translational modifications including decreased phosphorylation of HuR and an increased phosphorylation of AUF1. These changes may promote the dissociation of ζ-crystallin from the RNA-binding complex. This remodeling of the ribonucleoprotein-complex blocks the association of deadenylases and maintains a poly-A tail that is well protected by the poly-A binding protein (PABP). Therefore, this remodeling of the protein/mRNA complex mediates the enhanced stabilization and translation of PEPCK mRNA.
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    Small RNA methylation in Caenorhabditis elegans
    (Colorado State University. Libraries, 2020) Svendsen, Joshua, author; Montgomery, Taiowa A., advisor; Wilusz, Carol J., committee member; Nishimura, Erin Osborne, committee member; LaRocca, Thomas J., committee member
    Eukaryotes employ small RNAs and RNA interference (RNAi) for such diverse tasks as regulating gene expression, suppressing viral infections, and defending their genomes from foreign or parasitic sequences such as pseudogenes or transposons. In the germlines of animals, piwi-interacting RNAs (piRNAs) target and silence sequences such as these to ensure that genetic information passes from one generation to the next intact. For many animals, piRNAs must be methylated at their 3' ends to protect them from degradation, and loss of stable piRNAs results in sterility. In this study, we identify a role for piRNA methylation in maintaining germline integrity in C. elegans. We also show that methylation is important for piRNA stability in worm embryos but dispensable in the adult germline. Further, we identify additional classes of methylated small RNAs: exogenous primary short interfering RNAs (siRNAs) and a subpopulation of micro RNAs (miRNAs), as well as characterizing factors that influence which small RNAs are methylated. Finally, we provide a brief review of recent advances in the field of piRNA biology that detail the rules governing piRNA targeting and a means by which endogenous C. elegans genes avoid silencing by the piRNA-dependent RNAi pathway.