Doctor of Philosophy (PhD), Ohio University, 2022, Chemistry and Biochemistry (Arts and Sciences)

Antibiotic resistance is a global threat beside the ongoing pandemic by SARS-CoV-2. The number of deaths due to antibiotic-resistant infections is increasing at an alarming rate. The COVID-19 pandemic has already claimed millions of deaths worldwide. Fighting against antibiotic-resistant superbugs and the SARS-CoV-2 has become a challenge. A significant amount of research is going on to develop the vaccine and small molecule antiviral and antibacterial therapeutics targeting proteins. Fortunately, novel non-coding regulatory RNA targets have been identified for developing new antibacterial and antiviral drugs such as bacterial T-box riboswitch, RNA thermometers, and viral stem-loop II motif. T-box riboswitch can control the transcription or translation of amino acid-related genes in bacteria by forming unique interactions between tRNA and mRNA. RNA thermometers (RNATs) are temperature-responsive riboswitches that control the translation based on temperature sensing thus controlling the interaction with the mRNA and 16S rRNA. In Shigella dysenteriae, three RNATs, i.e., ompA, shuT, and shuA, have been discovered. ompA RNAT controls the translation of outer membrane protein A. shuT, and shuA RNAT controls the translation of two proteins that are crucial to the bacterial heme utilization system. The Stem-loop II motif (S2M) is a highly conserved RNA element found in most coronaviruses, astroviruses, and picornaviruses that plays a potential role in viral replication and invasion. The RNA structure plays a significant role in its regulatory function for all of these potential therapeutic targets. Consequently, it is essential to examine the factors that affect the RNA structure and RNA-RNA interaction. Despite having limited building blocks, RNA has diverse functions in the cells. Base protonation and protonated base pairs often occur in RNA when interacting with other biomolecules, thus could play a critical role in vital biological processes. Diff (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2019, Pharmaceutical Sciences

Ribonucleic acid (RNA) nanotechnology is a rapidly emerging field that focuses on the nanostructure design, construction, and application in biotechnology and biomedicine. Unlike other biomacromolecules, RNA is more flexible in structure and more versatile in functionality. On the other hand, RNA is a naturally-occurring biopolymer, making them more biocompatible compared to other nanomaterials. Thus, RNA can serve as a building block to construct nanoparticles as drug delivery platform for cancer therapy. This dissertation primarily describes a fundamental study that explores the immune-compatibility of RNA nanoparticles, as well as the use of thermostable RNA nanoparticles to specifically deliver therapeutics for efficient cancer therapy without causing toxicity. Firstly, RNA polygons that have identical size but varying shapes, or same shape but with different sizes were constructed as study model. The RNA nanoparticles were found to be immunologically inert, indicating that RNA nanoparticles are safe drug carriers without triggering immune responses. On the other hand, they can elicit significant immunomodulation by extending the nanoparticles with special sequences. Specifically, this immunomodulation was found to be size, shape, sequence-dependent, demonstrating the potential of using RNA nanoparticles in immunotherapy. Secondly, the use of a thermostable RNA nanoparticle for solubilizing and high-density loading chemotherapeutic drugs for cancer inhibition is reported. Small chemotherapeutic drugs possess significant anti-cancer activity, but their clinical applications were greatly limited by the poor biocompatibility such as water-insolubility. By chemically conjugating water-insoluble drugs to RNA, the RNA nanoparticles dramatically improved drug water-solubility. An ultra-thermostable RNA four-way junction nanoparticle was able to covalently load twenty-four copies of paclitaxel without nanoparticle dissociation or unfolding. After intravenous administrat (open full item for complete abstract)

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2012, Biological Sciences

Many hairpin and internal RNA 3D motif structures are recurrent, occurring in various types of RNA molecules, not necessarily homologs. Although usually drawn as single-strand “loops” in RNA 2D diagrams, recurrent motifs share a common 3D structure, but can vary in sequence. It is essential to understand the sequence variability of RNA 3D motifs in order to advance the RNA 2D and 3D structure prediction and ncRNA discovery methods, to interpret mutations that affect ncRNAs, and to guide experimental functional studies. The dissertation is organized into two parts as follows. First, the development of a new online resource called RNA 3D Hub is described, which is intended to provide a useful resource for structure modeling and prediction. It houses non-redundant sets of RNA-containing 3D structures, RNA 3D motifs extracted from all RNA 3D structures, and the RNA 3D Motif Atlas, a representative collection of RNA 3D motifs. Unique and stable ids are assigned to all non-redundant equivalence classes of structure files, to all motifs, and to all motif instances. RNA 3D Hub is updated automatically on a regular schedule and is available at http://rna.bgsu.edu/rna3dhub. In the second part of the dissertation, the development of WebFR3D (http://rna.bgsu.edu/webfr3d), a new webserver for finding and aligning RNA 3D motifs, is described and its use in a biologically relevant context is then illustrated using two RNA 3D motifs. The first motif was predicted in Potato Spindle Tuber Viroid (PSTVd), and the prediction was supported by functional evidence. The second motif had previously been undescribed, although it is found in multiple 3D structures. RNA 3D Hub, RNA 3D Motif Atlas, and the bioinformatic techniques discussed in this dissertation lay the groundwork for further research into RNA 3D motif prediction starting from sequence and provide useful online resources for the scientific community worldwide.

Doctor of Philosophy, Case Western Reserve University, 2024, Biochemistry

Gene expression in higher eukaryotic cells involves carefully orchestrated interactions between RNA-binding proteins (RBPs) and RNAs. Understanding the rules that govern the interactions between RBPs and RNAs is a central question in biology, and increasingly, in medicine. This thesis investigates the cellular RNA- RBP interactions in two ways. First, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RNA-binding protein HuR. Using this new method, we uncover HuR's unique binding preferences in the nucleus, cytoplasm, and stress granule, during mock and arsenite stress conditions. Second, using available datasets that describe RBP binding in vitro and in cells, we devise two quantitative metrics to directly compare RBP binding behaviors: inherent specificity and mutational sensitivity. These new metrics provide a new quantitative framework to characterize the binding behaviors of an RBP.

PHD, Kent State University, 2024, College of Arts and Sciences / Department of Chemistry and Biochemistry

An increase in the production of reactive oxygen species (ROS) and the inability of cellular machinery to adequately neutralize the ROS results in cellular oxidative stress. ROS can damage all major classes of biomolecules, proteins, lipids, DNA and RNA. Several studies including recent studies from our lab demonstrated the link between RNA oxidation and neurodegenerative disease, such as, Parkinson's Disease (PD) and Multiple Sclerosis (MS). In our recent study, selectively oxidized NAT8L mRNA and its cognate protein reduction were identified as major contributing factors that can be attributed to demyelination in MS. In addition to these findings, we have observed the presence of a non-canonical secondary structure G-quadruplex (GQ) forming sequence (PQS) on the coding region of NAT8L mRNA. Working on the NAT8L PQS segment, we discovered that it's oxidation destabilized the GQ adoption in low salt condition, whereas the same RNA sequence can overcome the effect of oxidation and form an equally stable GQ as its wild type under physiologically relevant salt condition (100 mM K+ ). Using mutational analysis, we have shown that the occurrence of the spare Gs in two out of four G-runs in NAT8L PQS, facilitates the participation of alternative Gs to adopt GQ structure when specific Gs get oxidized. A key unresolved question is the effect of 8-OG modification on RNA structure. To address that issue we embarked upon a study to locate and study the impact of 8-OG on the structurally well characterized Tetrahymena group I intron and its independently folding domain P4-P6. Based on our RNaseT1 structural mapping data, nearly 40% of the Gs in P4-P6 RNA get oxidized under oxidative microenvironment. Among them nearly 17% of the Gs are significantly oxidized (fold change in RNaseT1 protection ≥ 5). Interestingly, all of these Gs (G147, G180-G181, G188, G141, G163-G164) are located in the P5abc subdomain of P4-P6 and are known to be involve (open full item for complete abstract)

Bachelor of Science (BS), Ohio University, 2022, Biological Sciences

Analyzing host cell response using RNA-seq allows us to understand what is happening within the host cell that could be missed by narrower analysis. From the analyses in this study, we can broadly state that extracellular vesicles (EVs) produced by Staphylococcus aureus at different physiological temperatures have varying effects on host cell gene expression. Infection and immune system-related genes appear to be upregulated in THP-1-derived macrophages treated with EVs produced at 37°C (core body temperature). These genes then appear to be downregulated in macrophages treated with EVs produced at 34ºC (the temperature of the anterior nares, where S. aureus colonizes). This pattern observed with EV-treated macrophages was not observed in EV-treated human nasal epithelial cells (HNECs). By utilizing RNA-seq we can see on a global level how RNA introduced to human cells, via EVs or a future vaccine, has an effect on our own gene expression.

PHD, Kent State University, 2018, College of Arts and Sciences / Department of Chemistry

The piwi interacting RNAs (piRNAs) are small non-coding RNAs with mostly 24-32 nucleotides in length. The piRNAs are defined by their ability to specifically bind to the PIWI proteins, a requirement for their functions. The piRNAs are involved in germline development, transposon control, transcriptional and post-transcriptional gene regulation. However, piRNA mediated post-transcriptional gene regulation in human somatic cells is not well understood. We discovered a human piRNA (piR-FTH1) that negatively regulates the FTH1 expression at the post-transcriptional level in MDA-MB 231 triple negative breast cancer cells. Furthermore, we established that piR-FTH1 knocks down the FTH1 mRNA through the piR-FTH1-HIWI-RISC pathway by using the HIWI2 and HILI proteins. We determined that FTH1 repression by piR-FTH1 increases the sensitivity of the chemotherapeutic agent Doxorubicin by a remarkable 20-fold. In a related project, we investigated the role of piRNA structure in its function. Interestingly, the piRNAs do not have any defined secondary structure. Using bioinformatics analysis, we discovered the presence of putative G-quadruplex (GQ)-forming sequences in human piRNAs that are higher in number compared to the piRNA pools of other organisms that were analyzed. Using one GQ forming human piRNA sequence (piR-48164), we showed that formation of GQ structure in the piRNA led to inhibitions of the PIWI protein binding and target gene silencing in vitro and in cellulo. These studies unraveled the role of a non- canonical secondary structure GQ in the piRNA function and added a new layer of regulation in piRNA function. It has been reported previously that after the PIWI protein degradations, piRNAs become unprotected and undergo elimination. Prior to this study, the degradation mechanism of piRNA is unknown. We found that presence of 3' methylation of piRNA prevents its degradation through the exosome mediated decay pathway. We established that XRN1 and XRN2 are two 5' (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

Eukaryotic gene expression depends on the 5' cap structure added to all mRNAs. The biology of RNA capping is more dynamic than originally thought, with cytoplasmic recapping enabling spatial and temporal control of translation and other aspects of the RNA life cycle. Despite progress over the past decade, critical gaps remain in our understanding of the biochemistry of RNA recapping and its context within the cell, which are addressed in this dissertation. Recapping an uncapped RNA in the cytoplasm begins with the combined activities of a 5' monophosphate RNA kinase and capping enzyme (CE). Recapped RNAs are also methylated at the N7 position of the cap guanine, enabling recognition by cap-binding proteins such as the translation initiation factor eIF4E. However, it was unknown how caps synthesized in the cytoplasm acquire this critical methyl group. Here I describe the identification of the enzyme that completes the synthesis of mature caps in the cytoplasm. This enzyme, RNA guanine-7 methyltransferase (RNMT), was originally thought to be restricted to the nucleus, but I show that it also functions in the cytoplasm. Cytoplasmic RNMT activity is unexpectedly robust compared to that of nuclear RNMT, and RNMT knockdown points to RNMT being the predominant, if not only cap guanine-N7 methyltransferase in the cytoplasm. These results were obtained using an adapted cap methyltransferase activity assay, the details of which are provided. RNMT directly interacts with CE through its C-terminal catalytic domain, allowing it to associate with the multifunctional cytoplasmic capping complex. Cytoplasmic RNMT activity is additionally stimulated by dimerization with the small protein cofactor RAM. Inhibiting cytoplasmic cap methylation with a dominant-negative form of RNMT caused recapping target RNAs to destabilize, suggesting surveillance by decapping enzymes specific for unmethylated caps. The full complement of proteins required for cytoplasmic recapping is also unknow (open full item for complete abstract)

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2017, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Macrophages are critical cells in the immune system implicated in various diseases with a chronic inflammatory component, such as atherosclerosis. The dominant macrophage types, M1 and M2, play key roles in progression and regression of atherosclerotic plaques. In this study, we profiled the transcriptomes of these macrophage subsets using RNA-Seq and microarray to examine molecular signatures and distinctive pathways for each subset. RNA-Seq analysis revealed a total of 2,127 differentially expressed genes (including coding and non-coding transcripts) between the M1 and M2 subsets. Validation by qRT-PCR of 10 top differentially expressed genes showed that 8 of the upregulated and 9 of the downregulated genes followed the expected trend based on the RNA-Seq analysis. Subsequently, pathway analysis of the upregulated and downregulated gene sets showed M1 and M2 macrophages to be enriched in pathways such as the Th1 and glioma signaling respectively. Microarray analysis revealed a total of 163 differentially expressed circRNAs between the two macrophage subsets and predictions of circRNA/miRNA interactions. From these circRNA/miRNA interactions, a number of the differentially expressed circRNAs were predicted to target athero-relevant miRNAs. As examples, circRNA_41878 (gene Ankrd42) was predicted to target miR-382-5p while circRNA_19794 (gene Lmbrd1) was predicted to target miR-124-3p, miRNAs of recognized effects in atherosclerosis. Overall, this work provides insight into distinctive molecular signatures and pathways enriched in bone-marrow derived M1 and M2 macrophages, and reveals the presence and contribution of non-protein-coding RNAs to their transcriptomes. In addition, our circRNA results provide the basis for future studies on the function of circRNAs in the context of macrophage functions in atherosclerosis.

Doctor of Philosophy, The Ohio State University, 2017, Molecular Genetics

RNA-directed DNA methylation (RdDM) plays a key role in transcriptionally silencing both transposons and DNA viruses in many organisms. In plants, siRNAs guide ARGONAUTE (AGO)-containing silencing complexes to target DNA, which results in repressive histone and DNA methylation and subsequent heterochromatin formation. This silencing is critical for genome stability and antiviral defense. In canonical, or Pol IVRdDM, RNA Polymerase IV (Pol IV) generates non-coding RNA that is rendered doublestranded and processed into 24 nt siRNAs, which guide AGO4 to RNA Polymerase V scaffold transcripts. While regions of Pol IV and Pol V activity have been identified, significant information about their primary transcripts is lacking, including transcription initiation sites and possible promoter regions, 5' and 3' modifications, and whether the transcripts undergo splicing. In this work, we describe the characterization of Pol IV and Pol V-dependent non-coding transcripts generated from the intergenic region of Beet curly top virus. Additionally, we have identified non-coding transcripts that are Pol IV and Pol V independent, suggesting a role for Pol II in RdDM.

PHD, Kent State University, 2016, College of Arts and Sciences / Department of Chemistry

G-quadruplexes (GQs) are secondary structures that can be adopted by both DNA and RNA in the presence of monovalent cations and they are known to play important regulatory roles in a wide range of biological processes, such as, transcription, translation, splicing, mRNA localization, and telomere homeostasis. Although the functional roles of GQ structures have been extensively studied, the evolution of GQ structure is poorly understood. Thus, we investigated the evolutionary selection of GQ structures within the codon region (CDS) of mRNAs in a wide range of species and observed that the stable GQs are selected against within CDSs by synonymous codon usage. Furthermore, our results revealed context-dependent codon bias against the formation of stable GQs at the codon level resolution, thus delineating the evolutionary mechanism of GQ selection. Although a myriad of studies has reported the regulatory roles of GQs that are present in mRNAs, very little is known about the prevalence and functions of GQs in non-coding RNAs. We revealed the presence of GQ structures in precursor microRNA (pre-miRNA) and showed a regulatory role for GQs in miRNA biogenesis. Using a clinically important miRNA (miR-92b), we showed the existence of an equilibrium between GQ and the canonical stem-loop conformation which can regulate various stages of miRNA biogenesis such as nuclear export and Dicer-mediated maturation. Furthermore, we demonstrated that GQ structures can potentially be targeted in pre-miRNAs to treat diseases in which corresponding miRNAs are overexpressed. We showed that a rationally designed locked nucleic acid (LNA) oligonucleotide, which specifically binds with the GQ confirmation containing pre-miRNA can be used to treat non-small cell lung cancer where miR-92b is overexpressed. Although many different GQ binding ligands have been developed as therapeutics, the selectivity of these ligands is crucial to estimate the off-target effects. We developed an in vitro li (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2015, Physics

Interactions between RNAs and RNA-binding proteins (RBPs) are significant in post-transcriptional regulation. In this process, an mRNA molecule is bound by many proteins and/or microRNAs to modulate its function. It is therefore an interesting question how these multiple RBPs collaborate to enable combinatorial gene regulation. Here, we propose a possible mechanism which can support this RBP-RBP collaboration, termed "cooperativity". Such a cooperativity can exist merely based on fundamental principles of statistical physics and thermodynamics of RNA structure folding, without considering any further details of RNA and RBP properties. The theory is based on the idea that a successfully binding RBP will prohibit the formation of some originally allowed RNA structures, thus changing the statistical properties of the RNA structure ensemble, as well as the binding probabilities of other RBPs on the same RNA. In addition, this mechanism does not require direct physical interactions between RBPs, and thus supports the long-range characteristic of the cooperativity. Focusing on an RNA with two binding sites, we first calculate the correlation function between the RBPs on the RNA-RBP complex, verifying that this cooperativity exists. We then derive a characteristic difference of free energy differences, i.e. delta delta G, as a quantitative measure of this structure-mediated cooperativity. We apply this measure to a large number of human mRNAs, and discover that this cooperativity is a generic feature. Interestingly, this cooperativity not only affects binding sites in close proximity along the sequence but also configurations in which one binding site is located in the 5'UTR and the other is located in the 3'UTR of the mRNA. Some intriguing interplays between RBPs, microRNA binding sites, and UTR sequences are also disclosed. In the last chapter, we extend our model to handle multiple sequence-specified protein binding sites. We apply this extended model to the binding rea (open full item for complete abstract)

Bachelor of Science (BS), Ohio University, 2014, Biological Sciences

Shigella bacteria cause 165 million annual cases of Shigellosis, resulting in 1.1 million fatalities each year. With growing antibiotic resistance and no vaccine, new methods must be developed to treat Shigellosis. Outer membrane protein A, or OmpA, is required for Shigella virulence and may be a good target for inhibitory drugs. Before these new treatments can be developed, we need to understand the regulation of ompA and its role in virulence. In a previous study, it was found that ompA mRNA (the molecular template for making protein) levels in E. coli are decreased at higher temperatures while OmpA protein levels remain the same. This suggests that translation efficiency is affected by temperature dependent regulation. Protein is more efficiently made from the lesser amount of mRNA present at higher temperatures. This project determined that S. dysenteriae ompA has a similar temperature-dependent regulation. Future studies will be conducted to determine if this temperature dependent-regulation is mediated by a putative FourU RNA thermometer in the 5’ UTR of ompA. Additionally, future studies will examine the contribution of this putative RNA thermometer to S. dysenteriae virulence.

Doctor of Philosophy, The Ohio State University, 2006, Physics

RNA is crucial in life through both of its aspects, sequence and structure. In the beginning of this thesis, we study the statistical mechanics of RNA secondary structure formation. We first analytically describe the finite size effects. The crossover length, beyond which RNA stays in the thermodynamic limit, can be much longer than natural RNAs depending on the sequence pattern. Thus, one needs to consider the finite size effects whenever numerical or experimental results are to be interpreted within the framework in the thermodynamic limit. While this result holds only for homogeneous sequences, it might also hold for disordered RNA sequences. Studying disordered RNA is itself a very interesting challenge in statistical mechanics. A solid analytical description is still lacking due to the difficulty in calculating the quenched average. An alternative approach, called annealed averaging, is more tractable. However, the differences between annealed and quenched averaging can be very large. We quantify these differences, which are then used to improve the annealed system. The resulting constrained annealed system can predict certain thermodynamic quantities very close to the quenched results. We then switch to the study of RNA sequence as a genetic information carrier. An RNA sequence may undergo certain editing that can significantly alter its genetic information. Thus, usual computational approaches for gene search do not work when RNA editing occurs. We modify an algorithm to include the effects of editing, and predict three genes that have never been discovered before. Besides gene search, we also study certain feature of the editing sites. Editing in many organisms usually occurs with a certain bias in choosing the editing positions. Currently, this position bias is a mystery since a clear editing mechanism is lacking. We propose an evolutionary model that quantitatively explains the position bias of editing in the organism Physarum . This suggests that in Physa (open full item for complete abstract)

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2011, Biological Sciences

Base triples are recurrent sets of three interacting RNA nucleotides that hydrogen bond to each other along their base edges. Base triples occur frequently in structured RNAs, usually as parts of recurrent structural motifs or of tertiary interactions between parts of the RNA that are distant in the secondary structure. In almost all base triples, the central base interacts with each of the other bases of the triple to form two base pairs. As described in previous work by Leontis and Westhof, RNA nucleotides pair along their Watson-Crick (W), Hoogsteen (H), or Sugar (S) base edges and the resulting base pairs can be classified by identifying the interacting edges and the mutual orientations of the glycosidic bonds of the interacting nucleotides. The main goal of the present work is to test the hypothesis that the geometric base pair classification of Leontis and Westhof can be extended in a natural way to classify base triples geometrically. To test this hypothesis, the research leveraged the large number of RNA molecules that have been solved to atomic resolution by x-ray crystallography and deposited in the Protein Data Bank (PDB). Using a non-redundant (NR) data set of atomic-resolution, RNA-containing x-ray structures, a comprehensive survey of base triples was carried out in parallel with a combinatoric enumeration of base triple families predicted on the basis of the 12 geometric base triple families defined by the Leontis-Westhof nomenclature. This work predicts 108 potential geometric base triple families. Comprehensive searching of atomic-resolution RNA 3D structures found instances of 68 of the 108 predicted base triple families. Model building was carried out to determine which of the remaining 40 families are sterically feasible. Significantly, the proposed classification of base triples accounts for all but a handful of base triples observed in the structure database. The exceptions are intermediate cases in which two bases form a Watson-Crick pair an (open full item for complete abstract)

Master of Science (MS), Bowling Green State University, 2006, Chemistry

The application of rapid prototyping (RP) to the field of molecular modeling is growing due to the availability of computer programs and RP machines at major research institutions. Two RP techniques that are applicable to the study are powder-binder printing and Fused Deposition Modeling (FDM). Both of these technologies are available at BGSU and can be used in order to study the basepair interactions in many different types of RNA motifs. I have determined that the powder-binder technique is preferred when the tertiary structure of RNA is desired; conversely, FDM is better when the primary and secondary structures of RNA motifs are desired. By using FDM modeling, you can see the orientation of non-canonical basepairs, hydrogen bonds between atoms, the phosphate- sugar backbone, as well as structural motifs in RNA. I then applied our knowledge of RP technology by creating physical models of the C-loop motif, RNase P RNA, the kink-turn, the sarcin-ricin loop, and the SARS virus genome.

Master of Science, The Ohio State University, 2007, Graduate School

Doctor of Philosophy (PhD), Ohio University, 2024, Plant Biology (Arts and Sciences)

Plants encounter various biotic and abiotic stresses daily and have developed defense mechanisms to overcome these challenges. One key system involved in these defense mechanisms is the ubiquitin (Ub)-26S proteasome system (UPS), which targets malfunctioning proteins for degradation through Ub-tagged proteasomal pathways. The E3 ligases, specifically S-phase kinase-associated protein 1 (SKP1), Cullin 1 (CUL1), and F-box (SCF) complexes, play crucial roles in this process by recognizing and tagging specific protein substrates. Arabidopsis thaliana, with over 700 F-box proteins, has the largest group of E3 ligases, yet only 5% have been functionally characterized. Phylogenetic relationships among 111 plant species have identified four clusters of F-box genes, including a cluster with more conserved F-boxes, referred to as core Arabidopsis F-box (CAF) genes. Given that CAF genes have more known functions compared to other clusters, this dissertation hypothesizes significant potential for discovering new functions among the uncharacterized F-boxes within this group. Considering the evolutionary conservation of most CAFs, I adopted a genetic approach to investigate the roles of CAFs during seed germination and seed development. To address the challenges posed by functional redundancy of duplicated CAF genes and the lethality associated with constitutive F-box overexpression in transgenic plants, I created a library of inducible overexpression lines for 40 CAF genes, many of which lacked known biological functions. By systematically examining the effects of conditional overexpression of these 40 CAFs, I found that CAF overexpression during seed germination and seed development can positively or negatively regulate radicle rupture growth, thus controlling the germination process. Specifically, I identified 24 CAFs that enhance radicle rupture and two that inhibited it by interfering with abscisic acid (ABA)-mediated germination suppression. Induction of CAFs during seed (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2024, Biochemistry

Eukaryotic mRNA metabolism is a multifaceted process dictating diverse and essential cellular functions governing the regulation of gene expression. Central to mRNA metabolism, mRNA translation and decay play a pivotal role in regulation of gene expression. An outstanding question in the RNA field is whether translating and non-translating mRNAs exhibit similar decay rates. We studied the effects of current decay methods on translating mRNA and found that most decay methods lead to translational repression. To address this, we optimized a SLAMseq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA) protocol with a 4sU pulse-chase strategy. SLAMseq allows for parallel quantification of newly synthesized and existing RNA by directly tracking sites of 4sU labeling by monitoring T-to-C conversions in RNAseq data. The effect of 4sU and Uridine on translation was reduced by optimizing 4sU pulse duration and uridine chase concentrations. We employed our optimized SLAMseq protocol with a 4sU pulse-chase strategy, isolating total, monosomal, and polysomal RNA pools. We calculated mRNA half-lives for roughly 8,000 genes in the total mRNA pool and approximately 6,000 genes in monosomal and polysomal pools under two pulse-chase regimes. Notably, we observed significant variations in decay rates between these RNA pools. Gene level differences between our two pulse-chase regimes illustrate how instantaneous kinetic changes alter gene expression and the underlying complexity of kinetic regulation within the cell. Taken together, our data demonstrates a new approach to monitor translation stability providing insight into how mRNA lifespan diverges across different translational states, and emphasizes the importance of gene-level analysis for a comprehensive understanding of mRNA decay. These findings offer a method to study differences between co-translational and translation-independent decay rates.

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

Although impressive progress has been made to reduce the burden of heart disease, the illness remains the leading cause of death in the world. Notably, many types of persistent heart injury can cause pathological remodeling of the heart and progression to heart failure. The overall goal of my dissertation is to dissect novel molecular pathways responsible for such transition, since targeting them therapeutically presents an exciting opportunity to slow down heart remodeling and improve survival. While transcriptional regulators of progression to heart failure have been studied extensively, the importance of post-transcriptional regulation, such as chemical modification of messenger RNA, has been overlooked. Recent critical studies from our group and others demonstrated that methylation of mRNA in position N6 of adenosines (m6A) was essential for the heart's ability to adapt to stress, but the downstream regulators of this mechanism have remained elusive. Moreover, the field lacked a clear understanding of which cardiac m6A targets are most relevant and how they are regulated. After methylation, m6A-modified mRNAs are recognized by specific mRNA-binding proteins belonging to the YTH domain family (YTHDF), of which YTHDF1 and YTHDF2 are two key members expressed in the heart. In my work, I examine their specific contributions to the regulation of cardiomyocyte biology and their mechanistic effects on the distinct cardiac mRNA transcripts. First, I investigate the role of YTH N6-Methyladenosine RNA Binding Protein 2 (YTHDF2) in the heart using an inducible loss-of-function mouse model. I find that YTHDF2 protein is elevated in the failing mouse hearts and is essential for maintenance of cardiac homeostasis. Loss of YTHDF2 drives cardiac hypertrophy, fibrosis, and dysfunction in mice in the absence of any apparent stress. Furthermore, I detect that the proteome of YTHDF2-null cardiomyocytes is significantly remodeled, corroborating the importance of YTHDF2 in the re (open full item for complete abstract)