Browsing by Author "Wilusz, Carol, committee member"
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Item Open Access Factors and mechanisms of archaeal transcription termination and DNA repair(Colorado State University. Libraries, 2022) Marshall, Craig, author; Santangelo, Thomas J., advisor; Peersen, Olve, committee member; Wilusz, Carol, committee member; Yao, Tingting, committee memberRNA synthesis by RNA polymerase (RNAP) is an essential process and must be properly regulated both temporally and spatially to ensure cellular health in dynamic environments. Regulation of RNA synthesis in response to internal and environmental stimuli is typically achieved through interactions with RNAP at all stages of the transcription cycle- initiation, elongation, and termination. While studies of transcription initiation and elongation have identified multiple regulatory transcription factors and defined mechanisms, only a handful of protein factors able to terminate transcription have yet been described, and the general mechanism of transcription termination is still highly debated. We previously identified the first two factors capable of terminating transcription elongation complexes (TECs) in Archaea from the genetically tractable Thermococcus kodakarensis, and use both factors as models to explore the molecular mechanisms involved in collapse of the TEC. The Factor that terminates transcription in Archaea (FttA), a close homolog of the human CPSF subunit CPSF73, is completely conserved throughout Archaea, and appears to act analogously to the bacterial termination factor Rho, terminating transcription after the uncoupling of transcription and translation at the end of protein coding genes. We employed a novel genetic screen to verify the role of FttA in the polar repression of transcription, a phenomenon specific to regulation of genes contained within operons in prokaryotes. Eta, a euryarchaeal-specific superfamily 2 (SF2) helicase, appears to terminate transcription in a more specialized context, potentially terminating transcription of TECs arrested at sites of DNA damage while concurrently recruiting appropriate DNA repair enzymes, akin to the bacterial termination factor Mfd. A structure-function study of Eta employing select mutations derived from a crystallographic structure was conducted to elucidate the Eta-TEC contacts and various activities of Eta required for Eta-mediated termination. Further, many efforts were directed at establishing a role of Eta as an archaeal transcription-repair coupling factor (TRCF), and while this was not achieved, a state-of-the-art next-generation sequencing based approach to monitor nucleotide excision repair (NER) and the sub pathway transcription-coupled repair (TCR) genome-wide was developed and verified in E.coli. The work in this dissertation adds valuable insight to multiple fields of research. First, exploration into the mechanism of Eta-mediated transcription termination reveals a potential shared susceptibility of core RNAP subunits to transcription termination while elucidating activities of SF2 helicases- enzymes which are ubiquitously distributed in multiple essential cellular pathways. Second, our genetic screen identifies FttA as the archaeal polarity factor, shedding light on functions of an ancestral factor indispensable in mammalian transcription termination pathways. Establishment of the novel RADAR-seq/RNA-seq measurement of NER genome-wide will likely prove instrumental in future studies of archaeal DNA repair, and potentially presents a new paradigm in research of eukaryotic-like NER by use of Archaea as a advantageous model organism.Item Open Access Inherent aerobic capacity and susceptibility to breast cancer development(Colorado State University. Libraries, 2018) Lutsiv, Tymofiy, author; Thompson, Henry J., advisor; Wilusz, Carol, committee member; Foster, Michelle T., committee memberPhysical inactivity is one of the risk factors for developing breast cancer. Aerobic capacity is an objective measure of an individual's activity behavior as physical exercise improves their ability to consume, transfer, and utilize oxygen. Variability in responses to the same physical exercise program led scientists to determine that there are two components of aerobic capacity—inducible and inherent. The latter became possible to study when two models with high (HIAC) and low (LIAC) inherent aerobic capacity were created. A number of studies conducted on these models showed that not only do these strains differ in their exercise performance but also in their susceptibility to disease. LIAC animals gain more weight and exhibit reduced fatty acid oxidation compared with their HIAC counterparts, especially on a high-fat diet. Based on these observations, my working hypothesis is that inherent aerobic capacity underlies an individual's metabolic flexibility. Metabolically inflexible cells exhibit increased glucose utilization, anabolic metabolism, as well as cell proliferation and survival. Interestingly, similar factors are also associated with carcinogenesis. HIAC animals appear to be metabolically more flexible on a systemic and cellular level than their LIAC counterparts. Additionally, our laboratory previously showed that LIAC animals exhibited greater incidence, multiplicity, and lower latency of breast tumors than HIAC upon carcinogen administration. To reveal the underlying mechanisms of their different carcinogenic responses, we analyze protein expression patterns in the mammary gland and tumors of HIAC/LIAC models. We demonstrate that LIAC animals upregulate pathways associated with glucose utilization, protein and fatty acid synthesis, as well as other carcinogenic signatures, whereas HIACs are associated with energy sensing, fatty acid oxidation, and cell cycle arrest. Consequently, we propose that higher inherent aerobic capacity renders cells metabolically more flexible and reduces their susceptibility to breast cancer development.Item Open Access Investigation into Mycobacterium bovis phenolic glycolipid as a potential biomarker of bovine tuberculosis in urine(Colorado State University. Libraries, 2019) Morphet, Stephanie Marie Little Thunder, author; Belisle, John, advisor; Crick, Dean, committee member; Wilusz, Carol, committee member; Henry, Chuck, committee memberMycobacterium bovis, the etiological agent of bovine tuberculosis (bTB) is reported to cause disease in man and animal alike on every continent aside from Antarctica. Although coordinated efforts have been made for over a century in the US to cease transmission of this pathogen, outbreaks still occur. It has been posited that the failure to eliminate transmission of this pathogen is partially due to the diagnostic in use, which lacks critical sensitivity and specificity. To address this gap, we investigated a potential new method of identifying infected animals that is through the detection of a pathogen-derived biomarker. M. bovis phenolic glycolipid (PGL) is a species-specific, highly abundant, and unique glycolipid that comprises up to 2.5% of the dry cell mass. Coupling an LC-ESI-TOF-MS, method with a solid phase extraction, we successfully detected PGL derived from the urine of naturally-infected cattle. With this knowledge, we aimed to generate a detector of PGL that could be applied in a rapid and field friendly diagnostic platform. Using phage display technology, we selected M13 bacteriophage capable of binding M. bovis PGL with specificity that differentiated between M. bovis PGL and M. Canetti PGL, as well as between total lipid fractions of various species of Mycobacterium and other lipids with similar biochemical properties to PGL. These M. bovis PGL specific phage were able to differentiate between unspiked cattle urine and urine spiked with PGL. Lastly, we assessed the relative stability of PGL, specifically contrasted to another highly abundant mycobacterial glycolipid, phosphatidylinositol mannoside (PIM). We found that PGL was the more stable molecule when testing thermal and chemical stability, as well as when treated with protease K. However, when treating these glycolipids with whole cell lysates derived from fresh bovine organs, accurate detection on an LC-ESI-TOF-MS platform was lost. Further studies will be required to probe the stability of these molecules in vivo. Overall, this potential methodology to assess for infection status may be beneficial in improving the control of M. bovis if further developed, as this new approach has the potential to be more specific and sensitive than the currently used diagnostic.Item Open Access Investigation of transcriptional dynamics in the Caenorhabditis elegans intestine gene regulatory network(Colorado State University. Libraries, 2022) Williams, Robert Thomas Patton, author; Osborne Nishimura, Erin, advisor; Wilusz, Carol, committee member; Hansen, Jeffrey, committee member; Santangelo, Tom, committee memberELT-2 is the major transcription factor required for the activation of Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos to promote development and persists after hatching through larval and adult stages. Though the sites of ELT-2 binding have been defined and the transcriptional changes that result from ELT-2 depletion described, the intestine-specific transcriptome profile over developmental time has not been characterized, in part because of the difficulty in isolating intestine from other tissues. To address this knowledge gap, we used Fluorescence Activated Cell Sorting (FACS) to enrich intestine cells and performed RNA-seq analysis at distinct developmental stages. By linking the transcriptome profiles to previous ELT-2 studies, we were able to gain new insight into the role of ELT-2 in the intestinal regulatory network throughout development. Correlation of ELT-2 binding to the intestine transcriptome data, revealed that only 33% of intestine-enriched genes were direct targets of ELT-2 binding in embryos, but that number increased to 75% by the L3 stage. This suggests additional transcription factors may promote intestine-specific transcription early in development. Consistent with this possibility, half of the ELT-2 direct target genes were not transcriptionally dependent on ELT-2 for appropriate expression (i.e. their expression was not impacted following ELT-2 depletion) implying that other factors may compensate in the absence of ELT-2. Among direct target genes that were affected by ELT-2 depletion, equal proportions were over and under-expressed thus ELT-2 can both activate and repress direct target genes. Both activated and repressed sets of ELT-2 target genes were enriched for defense response genes reinforcing recent findings demonstrating ELT-2 participating in mediating the immune response upon pathogen exposure. Fluorescent reporter assays demonstrated that expression of two direct targets of ELT-2 cebp-1 and ets-4 are indeed repressed by ELT-2. Moreover, we observed that ELT-2 repressed its own promoter in a negative feedback loop that regulates elt-2 gene expression. Together, our findings illustrate that ELT-2 contributes directly to roughly 20 – 50% of intestine-specific gene expression, that ELT-2 exerts both positive and negative regulatory control on its direct targets, and that our overall picture of the intestinal regulatory network is incomplete with more intestine specific transcription factors and mechanisms remaining to be discovered.Item Open Access Microbial succession in human rib skeletal remains and fly-human microbial transfer during decomposition(Colorado State University. Libraries, 2022) Deel, Heather Leigh, author; Metcalf, Jessica L., advisor; Wilusz, Carol, committee member; Trivedi, Pankaj, committee member; Pante, Michael, committee memberHuman decomposition is a dynamic process partially driven by the actions of microbes. It can be defined by the fresh, early decomposition, advanced decomposition, and skeletonization stages. The microbial communities that facilitate decomposition change in a predictable, clock-like manner, which can be used as a forensic tool for estimating postmortem interval. Chapter 1 introduces this concept by describing the stages of decomposition in detail and how high-throughput sequencing methods can be used with microbes to develop models for predicting postmortem interval. Chapter 1 also describes which sample types are most useful for predicting postmortem interval based on the stage of decomposition, the knowledge gaps in the field, and the steps necessary for adoption of this tool into the justice system. During fresh and early decomposition, microbial succession of the skin and soil sample types are most predictive of postmortem interval. However, after approximately the first three weeks of decomposition, the changes in the microbial communities that are used for predictions begin to slow down and the skin and soil sample types become less useful for estimating postmortem interval. Chapter 2 of this dissertation shows that microbial succession of the bone microbial decomposer communities can be used for estimating postmortem interval during the advanced and skeletonization stages of decomposition. First, the bone microbial decomposer community was characterized using 16S ribosomal RNA sequencing from six human donor subjects placed in the spring and summer seasons at the Southeast Texas Applied Forensic Science Facility. A core bone decomposer microbiome dominated by taxa within phylum Proteobacteria was discovered, as well as significant overall differences in the bone microbial community between the spring and summer seasons. These microbial community data were used to develop random forest models that predicted postmortem interval within +/- 34 days over a 1–9-month time frame of decomposition. To gain a better understanding of where the microbes in the decomposed bone were coming from, as healthy, living bone is typically sterile, SourceTracker2 was used with paired skin and soil samples taken from the same decedents. Results showed that the bone microbial decomposer community is likely sourced from the surrounding environment, particularly the skin and soil communities that occur during the advanced stage of decomposition. Chapter 3 of this dissertation focuses on the influence of the blow fly (Calliphoridae) microbiome on human cadaver microbial community assembly. In early decomposition, volatiles attract blow flies to the cadaver, which serves as a source of nutrients and a safe place to lay eggs. It is likely that during this interaction between hosts, there is a mechanical transfer of microbes that subsequently alters each of their microbial communities. While studies have shown that blow flies have their own microbiome, they were not conducted in a decomposition environment. First, Chapter 3 shows the characterization of the blow fly microbiome by organ and season in a terrestrial, human decomposition environment. This was performed by placing ten cadavers across the winter, spring, and summer seasons at the Southeast Texas Applied Forensic Science Facility, collecting the first wave of colonizing flies for each cadaver, and sequencing the 16S ribosomal RNA gene of the labellum (mouth parts), tarsi (leg parts), and oocytes. Results showed that the previously defined universal fly microbiome persists even in a decomposition environment, with notable differences still present between organs and seasons. Additionally, results from using the tool SourceTracker2 showed that the labellum and tarsi act as substantial bacterial sources of the human decomposer bacterial community, and this source contribution varies by season. In summary, this dissertation provides the first quantitative estimate of postmortem interval of terrestrially decomposed human skeletal remains using microbial abundance information. This is a significant contribution to the criminal justice system; anthropologists typically use visual evidence to provide postmortem interval estimates of skeletal remains with errors ranging from months to years, whereas our approach provides estimates with errors of approximately one month. Furthermore, this dissertation shows evidence that there is a mechanical transfer of microbes between blow flies and human cadavers during the early stage of decomposition, which provides ecological insight into human cadaver microbial community assembly.Item Open Access Post-initiation regulatory mechanisms of transcription in the Archaea(Colorado State University. Libraries, 2023) Wenck, Breanna Renée, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Osborne Nishimura, Erin, committee member; Wilusz, Carol, committee memberIncreasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. Archaeal systems contain simplified, basal regulatory transcription components and mechanisms homologous to their eukaryotic counterparts, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions. Many archaeal genomes are organized with histone proteins that resemble the core eukaryotic histone fold, which permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that impacts transcription regulation and gene expression. Despite such semblance between the eukaryotic and archaeal core histone folds, archaeal genomes lack the canonical N and C terminal extensions that are abundantly modified to regulate transcription in eukaryotic genomes. Much of what is known regarding factor-mediated transcription regulation in the Archaea is limited; however combined and continued efforts across the field provide tidbits of information, but many pieces are still missing. This thesis aims to i) delineate the role key residues within the histone-DNA complex and archaeal histone-based architecture and key residues within the histone-DNA complex have on the progression of the transcription apparatus, characterize factor-mediated transcription termination, and explore chromatin- and TFS-mediated regulatory effects on transcription via global RNA polymerase (RNAP) positions.Item Open Access Regulation of transcription by ubiquitin and the INO80 chromatin remodeling complex(Colorado State University. Libraries, 2015) Ndoja, Ada, author; Yao, Tingting, advisor; Cohen, Robert, advisor; Stargell, Laurie, committee member; Hansen, Jeffrey, committee member; Wilusz, Carol, committee memberTranscription in eukaryotes is a crucial process that is tightly regulated in order to maintain cellular homeostasis and offer optimal responses to environmental changes. Transcriptional activators (TAs) regulate this process by orchestrating time and locus-specific assembly of complex transcription machinery. Thus, the abundance, localization and activity of TAs are all subject to tight control. One way in which TAs are controlled is by the covalent attachment of the conserved protein ubiquitin. Ubiquitination of TAs has been reported to affect transcription via proteolytic and non-proteolytic routes, yet the function of the ubiquitin signal in the non-proteolytic process is poorly understood. Through studies of a series of synthetic and natural activators in yeast and mammalian cells, we found that mono-ubiquitinated TAs cannot stably interact with DNA to promote transcription. We have identified the AAA+ ATPase Cdc48 and its cofactors as the Ub receptor responsible for extracting mono-ubiquitinated activators from chromatin, independently of proteolysis. These findings describe a novel mechanism by which gene repression can be maintained without destroying the activator. Upon appropriate stimulus, deubiquitinating enzymes can readily reverse mono- or oligo-ubiquitination of the activator and transcription can initiate without the requirement for new protein synthesis. This mechanism may facilitate rapid switching between “on” and “off” states of transcription and may serve as a general strategy to prevent spurious transcription in the “off” state. Compaction of DNA into chromatin imparts further layers of complexity to the control of eukaryotic gene expression. Cooperation between chromatin remodelers, histone variants, and histone post-translational modifications (PTMs) offers diverse regulatory options in DNA metabolic processes, including transcription and DNA repair. The human INO80 chromatin-remodeling complex (hINO80) has been shown to facilitate transcription by promoting an open chromatin environment at promoter regions. How and whether hINO80 directly promotes an open chromatin environment is not yet understood. In an effort to elucidate how hINO80 regulates transcription, we have characterized the nucleosome sliding activity of hINO80 and examined how histone variant H2A.Z and histone PTMs modulate its activity in vitro. Our results suggest that nucleosomes containing H2A.Z or the H3 acetylation mimic, K56Q, are mobilized by hINO80 with faster kinetics compared to canonical unmodified nucleosomes, and their effects are additive. In contrast, ubiquitination of H2A or H2B does not affect the sliding activity of hINO80. Nucleosomes containing both H2A.Z and H3-K56Ac are enriched at promoter regions and DNA damage sites in mammalian cells. These nucleosomes have been shown to exhibit rapid turnover kinetics in vivo. Our studies provide biochemical evidence that hINO80 participates in transcription and DNA repair processes by ATP-dependent mobilization of H2A.Z/H3-K56Ac-containing nucleosomes. Future studies will be required to elucidate how nucleosome mobilization in vitro relates to chromatin dynamics in vivo.Item Open Access RNA replication by poliovirus RNA-dependent RNA polymerase: effects of residue 5 on elongation complex stability and processivity(Colorado State University. Libraries, 2011) Hobdey, Sarah Elizabeth, author; Peersen, Olve B., advisor; Stargell, Laurie, committee member; Chen, Chaoping, committee member; Wilusz, Carol, committee memberThe poliovirus (PV) RNA-dependent RNA polymerase (RdRP) is a small, single-subunit, enzyme that is responsible for the replication of the viral genome. PV genome replication is much more involved than just repetitively incorporating a single nucleotide into an oligonucleotide primer. The polymerase must first go through multiple steps of initiation before processive replication can happen. During primer-dependent initiation, the polymerase must bind the primer/template RNA substrate and undergo a conformational change to form a stable RNA-polymerase complex. After the stable RNA-polymerase complex is formed the polymerase undergoes a second conformational change associated with the addition of the first nucleotide to the primer. It is only after these few steps of initiation that the polymerase can begin processive elongation. The work presented in this dissertation addresses a structure-function relationship related to initiation and processivity of the PV RdRP. Specifically, my work shows that the PV RdRP residue 5 is involved in forming and maintaining the stable, elongation-competent complex. Also, the complex stability can be modulated by downstream RNA interactions or by the number of nucleotides that are incorporated to form the stable complex. Lastly, my data demonstrate evidence of an RNA rearrangement during elongation complex formation and show that the maintenance of a stable elongation complex is required for processive RNA replication, which is required for virus replication. To end, these data elucidate a more complete understanding of the structure-function relationships of the viral RNA polymerase and could eventually facilitate in the design of specific polymerase inhibitors.Item Open Access Stable kinetochore-microtubule attachment is sufficient to satisfy the spindle assembly checkpoint(Colorado State University. Libraries, 2016) Tauchman, Eric Cary, author; DeLuca, Jennifer G., advisor; Chen, Chaoping, committee member; Ross, Eric, committee member; Wilusz, Carol, committee memberDuring mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. The kinetochore proteins that bind spindle microtubules are exquisitely regulated to ensure correct segregation of genetic material at mitotic exit. Aurora B Kinase (ABK) phosphorylates Hec1, a protein that directly binds microtubules. This is critical for enabling the release of incorrect kinetochore-microtubule attachments. Hec1 has nine ABK phosphorylation sites on its tail domain allowing for precise control over binding affinity. We find that at least 7 of these sites are required for wild-type kinetochore-microtubule (K-MT) attachment stability as evaluated by inter-kinetochore distance measures and chromosome alignment capability. We further observe that several sites may have more influence on K-MT attachment stability than others. Hec1 mutations preventing phosphorylation increase kinetochore-microtubule attachment stability. In the absence of stable kinetochore–microtubule (K-MT) attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable Hec1mutant, a core component of the attachment machinery. This mutant reduced the ability of ABK to cause release of erroneous K-MT so we could test the hypothesis that stable K-MT attachments satisfy the SAC even if those attachments deviate from the canonical bipolar form. We find that stable attachments are sufficient to satisfy the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. These results indicate a conformational change(s), within the kinetochore that occurs upon stable kinetochore-microtubule binding causes the eviction of SAC proteins. This advance in our understanding of SAC function offers insight into the mode of action and the variation in cellular response to mitotic arrest therapies often used in treatments of cancers.Item Embargo 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.Item Open Access Translation-dependent mRNA localization in the Caenorhabditis elegans embryo(Colorado State University. Libraries, 2022) Winkenbach, Lindsay P., author; Osborne Nishimura, Erin, advisor; Wilusz, Carol, committee member; Stasevich, Tim, committee member; Di Pietro, Santiago, committee memberThough each animal cell contains the same genetic information, cell-specific gene expression is required for embryos to develop into mature organisms. Embryos rely on maternally inherited components during early development to guide cell fate specification. In animals, de novo transcription is paused after fertilization until zygotic genome activation. Consequently, early embryos rely on post-transcriptional regulation of maternal mRNA to spatially and temporally regulate protein production. Caenorhabditis elegans has emerged as a powerful developmental model for studying mRNA localization of maternally-inherited transcripts. We have identified subsets of maternal mRNAs with cell-specific and subcellular patterning in the early C. elegans embryo. Previous RNA localization studies in C. elegans focused on maternal transcripts that cluster in the posterior lineage and showed mRNA localization occurs in a translation-independent manner through localization sequence elements in the 3'UTR. However, little is known about the mechanisms directing RNA localization to other subcellular locales in early embryos. Therefore, we sought to understand the localization of maternal transcripts found enriched at the plasma membrane and nuclear periphery, erm-1 (Ezrin/Radixin/Moesin) and imb-2 (Importin Beta), respectively. In this thesis, I characterize two different translation-dependent pathways for mRNA localization of maternal transcripts at the plasma membrane and nuclear periphery. I identified the PIP2-membrane binding region of the ERM-1 proteins is necessary for erm-1 mRNA localization while identifying additional membrane localized maternal transcripts through the presence of encoded PIP2-membrane binding domains. Additionally, I observed that mRNA localization patterns can change over developmental time corresponding to changes in translation status. For imb-2 mRNA localization, I found localization to the nuclear periphery is also translation-dependent. Through recoding the imb-2 mRNA sequence while maintaining the translated peptide sequence using alternative codons, I found both localization and transcript stability additionally depends on mRNA sequence context. These findings represent the first report of a translation-dependent localization pathway for two maternally-inherited transcripts in C. elegans and demonstrate the utility of C. elegans as a model for studying translation-dependent mRNA localization during development.