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Theses and Dissertations

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Browsing Theses and Dissertations by Author "Abdo, Zaid, committee member"

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    The epitranscriptome in heat-loving Archaea enhances thermophily
    (Colorado State University. Libraries, 2023) Fluke, Kristin Alison, author; Santangelo, Thomas J., advisor; Wilusz, Carol, committee member; Sloan, Daniel, committee member; Abdo, Zaid, committee member
    >170 RNA modifications are known to decorate the transcriptome across all three Domains of life. The totality of RNA modifications in a cell is called the epitranscriptome. Modifications expand the form and function of RNA, often invoking new structures, activities, and interactions. The molecular consequences, fitness impacts, transcriptome-wide distribution, and genesis of the vast majority of modifications are largely unknown, but more > 100 human diseases are linked to mutations in the genes that encode RNA modifying enzymes. It is therefore critical to elucidate the generation and impact of RNA modifications on fitness and function. 5-methylcytidine (m5C) is one of the most abundant and conserved modifications across Domains and is generated through the post-transcriptional activities of several RNA m5C methyltransferases (R5CMTs). RNA modifications, especially m5C, have largely been studied in the context of abundant rRNA and tRNAs while research into the impact of mRNA modifications is lacking due to their low abundance in the cell. Archaeal model organisms have been shown to incorporate a higher abundance of select modifications compared to Eukarya, proving a new avenue to resolve fundamental questions regarding the phenotypic consequences of epitranscriptomic changes. In the model hyperthermophilic archaeon, Thermococcus kodakarensis, I comprehensively mapped m5C to the transcriptome. I identified at least five R5CMTs that site-specifically generate m5C and showed an unprecedented level of m5C incorporation that includes 10% of unique transcripts, mainly in mRNA. I demonstrated that R5CMTs target mRNAs for modification with both sequence and structural specificity. Cells lacking m5C exhibit a severe temperature dependent growth defect, indicating the m5C epitranscriptome is critical for cellular fitness under heat stress. The extensive m5C epitranscriptome coupled with the large collection of R5CMTs indicate that T. kodakarensis is the ideal model system to pursue fundamental questions regarding the epitranscriptome. Efforts to identify RNA methyltransferases that install m5C led to the discovery of a novel modification, N4,N4-dimethylcytidine (m42C) and the enzyme responsible for its in vivo and in vitro installation. I showed that m42C is robustly resistant to bisulfite-driven deamination, potentially indicating that all bisulfite-sequencing datasets may be falsely reporting m5C sites that are instead occupied by m42C. I mapped a single m42C residue to the ribosomal decoding center in the 16S rRNA and showed that cells lacking m42C exhibit a severe growth defect at higher temperatures. Structural studies of the enzyme that generates m42C, tentatively named m42C synthase, demonstrate it adopts a canonical class I Rossman fold at the C-terminal lobe and a unique N-terminal lobe. I showed that m42C synthase methylates assembled ribosomes and defined the catalytic amino acid residue. Taken together, I report a novel writer enzyme and show that both m5C and m42C promote hyperthermophilic growth. The dense and chemically diverse epitranscriptome argues that Thermococcus provides an excellent model system for further epitranscriptomic studies that probe the impact of both ubiquitous and rare modifications on core biological processes.
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    Time series analysis of limber pine (Pinus flexilis) health in the U.S. Rocky Mountains in response to white pine blister rust (Cronartium ribicola) and bark beetles
    (Colorado State University. Libraries, 2018) Leddy, K. A., author; Stewart, Jane E., advisor; Abdo, Zaid, committee member; Sloan, Dan, committee member; Schoettle, Anna, committee member; Liber, Howard, committee member
    From 2004-2007, 106 permanent limber pine monitoring plots were established and measured throughout the U.S. Rocky Mountains (MT, WY, CO) to characterize health trends in response to white pine blister rust (WPBR) and bark beetles (including mountain pine beetle, "MPB", and Ips spp., "Ips") over time. These plots were subsequently measured in 2011-2013 and again in 2016-17 to form a time series analysis of limber pine health. Data were gathered on 8,206 monumented trees (4,176 limber pine) and included measurements on various stand, ground cover, and landscape characteristics over the three time intervals. The overall percentage of live trees infected with WPBR was 29.4% in 2004-07 and 25.7% in 2016-17, with incidence decreasing in parts of Wyoming (Pole Mountain, Laramie Peak), increasing in southern Colorado (Sangre de Cristo Mountains), and stable in other subregions. However, of limber pines that were healthy during the first measurement, 22.2% were declining/dying and 21.1% had died by the end of the study period due to WPBR and/or bark beetle damages. Due to this, it is likely that new WPBR infections are occurring as the large number of live, infected trees dying during the survey may have masked newly infected trees in incidence calculations. In heavily WPBR-infected areas such as Pole Mountain, Wyoming, 65% of live trees were infected (in 2004-07), and of trees that began the study as healthy, 23% were declining or dying and 38% had died by the end of the study period (2016-17). Additionally, WPBR severity increased significantly from the beginning of the study with 4 previously uninfected sites gaining WPBR infections, 29 sites advancing to 'moderately infected' and 5 sites becoming 'heavily infected'. The overall average number of cankers per tree (3.5) was stable, but the number of infected limber pine with a canker in the lower 1/3 of the stem (18%) increased significantly (+4.2%, P = 0.001). When examining all limber pine in the study, 8%, 3% and 3% were killed by MPB/Ips., WPBR, and combined effects of these agents, respectively. Of the 887 live, but declining or dying limber pine, 52% had WPBR infections and 38% had damage from twig beetles (Pityophthorus spp., Pityogenes spp.) in 2016-17. Though all sites had ≥ 20% limber pine composition, 34% of sites had no limber pine regeneration and 7% had no regeneration of any tree species over the entirety of the study period. The results of this time series indicate that limber pine populations in the U.S. Rocky Mountains are declining due to effects from WPBR and MPB/Ips. Long-term surveys capture the effects of these damage agents on native tree populations and provide critical guidance for future management and restoration of these ecologically valuable species. Limber pine is at risk due to the various biotic and abiotic agents threatening their health. Thus, future directions involve restorative management practices for highly impacted areas where limber pine is a climax species and proactive management for healthy limber stands to promote resilience to likely damage agents. In highly impacted areas (WPBR incidence, mortality, or bark beetle damage on >50% of trees and low limber pine density and regeneration), where limber pine co-exists with other tree species, it may be favorable to allow the natural succession of other tree species to become dominant. However in xeric, harsh sites where limber pine is a climax species, these highly impacted areas are at-risk for losing all tree cover and should be considered for protective and restorative planting strategies. As natural resistance to WPBR occurs on the landscape, genetic screening and protection of mature limber pine carrying either complete or partial resistance to the pathogen should be pursued to preserve this genetic diversity. A priority should be to protect resistant against bark beetles and fire using established management practices. Additionally, seed-sourcing from resistant trees can allow for resistant progeny to be out-planted into high priority areas, thus buffering stands at risk for high WPBR mortality. Moreover management plans that promote diversification of age and diameter classes within stands can provide resilience against pest and pathogen attacks, as bark beetles vary in diameter preference and WPBR infections tend to cause higher mortality in smaller diameter trees. Lastly in healthy limber pine stands, proactive management of pest impacts to promote stand resilience is recommended as in Schoettle & Sniezko (2007) in order to preserve these healthy populations.