Doctor of Philosophy, The Ohio State University, 2016, Microbiology

RNA editing and RNA modification are chemical processes often essential for the survival of self-replicating organisms. RNA editing enables any RNA species to expand its decoding capacity or alter its coding potential using post-transcriptional enzyme catalyzed reactions. RNA modifications are equally important and can optimize the function as well stabilize many types of RNA. Trypanosoma brucei has provided ground-breaking discoveries in the field of RNA editing and modification which have gone against biological norms. A subset of tRNAs undergo adenosine to inosine (A to I) editing of their anticodon which permits decoding of up to three codons with a single tRNA. A few of these tRNAs are additionally edited from cytidine to uridine (C to U) in their anticodon stem at position 32 and further methylated at this same position. T. brucei possess two homologs of the 3-methylcytidine methyltransferase which catalyzes methylation of C or U at position 32. We have shown only one homolog is responsible for position 32 modification in this organism and my work has focused on the importance, and begun to elucidate, the biological function of the homologous 3-methylcytidine methyltransferase which we have named MTase37. Chapter 2 focuses on the importance of MTase37 for cytokinesis and ribosome stability and/or assembly. Using RNA interference (RNAi) I have determined MTase37 is specifically important for the stability and/or biogenesis of the large ribosomal subunit. MTase37 depletion causes loss of small ribosomal RNAs srRNA 4 and 5S from ribosomes. At the onset of an RNAi phenotype, cells show an increased number of flagella as well as increased nuclear and kinetoplastid DNA content. RNA processing is not affected in these cells, but we believe MTase37 plays an essential role in ribosome maturation acting as an RNA methyltransferase. In continuing to build upon our understanding of MTase37 Chapter 3 focuses on its modification activity and impact on cellular processes a (open full item for complete abstract)

Committee: Juan Alfonzo Ph.D. (Advisor); Patrice Hamel Ph.D. (Committee Member); Michael Ibba Ph.D. (Committee Member); Jane Jackman Ph.D. (Committee Member) Subjects: Biochemistry; Cellular Biology; Microbiology; Parasitology

PhD, University of Cincinnati, 2013, Arts and Sciences: Chemistry

The biogenesis and assembly of the ribosome involves a coordinated cascade of events including rRNA processing, folding, and post-transcriptional modifications of rRNA along with the association of ribosomal proteins (r-proteins). Unraveling the pathways and dynamics of these complex structural processes is a significant challenge. While conventional biophysical techniques such as nuclear magnetic resonance (NMR), X-ray crystallography and cryo-electron microscopy (cryo-EM) provide high-resolution information, these methods are not ideally suited to characterize transient and heterogeneous ribosome assembly intermediate structures. Here mass spectrometry-based approaches are being used to gain insights into the composition and structural organization of ribosomes and ribosome assembly particles in vivo, particularly those particles that result from perturbations (e.g. deletion of assembly factors, antibiotics). 15N-labeling and data-dependent LC-MS/MS were used to characterize the proteins associated with pre-30S complexes from E. coli RimM and RbfA deletion strains. RimM and RbfA are ribosome assembly factors implicated in the maturation of the small 30S subunit in bacteria. The precise roles of these assembly factors in 30S subunit assembly are unclear. Along with in vivo x-ray footprinting and mass spectrometry data, detailed molecular mechanisms how RimM and RbfA facilitate maturation of the 30S subunit in vivo were uncovered. Although relative quantitation of proteins by 15N-labeling and LC-MS/MS provides information on the differential expression of proteins in normal and perturbed samples, this approach is limited to comparing two samples at a time, labeling can be expensive and laborious, and not amenable to other multicellular organisms. The applicability of a label-free approach, LC-MSE for absolute "ribosome-centric" quantification of r-proteins was evaluated. Using an additional dimension of gas-phase separation through ion mobility and multip (open full item for complete abstract)

Committee: Patrick Limbach Ph.D. (Committee Chair); Albert Bobst Ph.D. (Committee Member); Joseph Caruso Ph.D. (Committee Member) Subjects: Analytical Chemistry

Doctor of Philosophy, The Ohio State University, 2020, Microbiology

In all organisms, protein synthesis is catalyzed by the ribosome, a large complex composed of RNA and proteins. The ribosome is assembled through multiple steps including transcription, cleavage and modification of rRNA, synthesis of ribosomal proteins, and the coordinated folding of RNA and proteins into mature particles. Ribosome assembly is complex and still poorly understood. A growing body of evidence suggests that GTPases play a critical role in ribosome assembly, and several conserved bacterial GTPases remain to be characterized. The physiological role of LepA, a paralog of EF-G found in all bacteria, has been a mystery for decades. Here, we show that LepA functions in ribosome biogenesis. In cells lacking LepA, immature 30S particles accumulate. Four proteins are specifically underrepresented in these particles—S3, S10, S14, and S21—all of which bind late in the assembly process and contribute to the folding of the 3' domain of 16S rRNA. Processing of 16S rRNA is also delayed in the mutant strain, as indicated by increased levels of precursor 17S rRNA in assembly intermediates. Mutation ΔlepA confers a synthetic phenotype in absence of RsgA, another GTPase, well known to act in 30S subunit assembly. Analysis of the ΔrsgA strain reveals accumulation of intermediates that resemble those seen in the absence of LepA. These data suggest that RsgA and LepA play partially redundant roles to ensure efficient 30S assembly. BipA is a conserved translational GTPase that resembles elongation factor EF-G and 30S assembly factor LepA. Recent evidence suggests that BipA functions in 50S subunit assembly, but the precise role of the factor remains unclear. During growth at suboptimal temperature, loss of BipA leads to accumulation of immature large subunit particles (~40S) that lack several proteins. These include L2, L14, L16, L17, L19, L28 and L32. Parallel analysis of the control (wild-type) strain shows accumulation of virtually identical intermediate parti (open full item for complete abstract)

Committee: Kurt Fredrick (Advisor); Michael Ibba (Committee Member); Karin Musier-Forsyth (Committee Member); Charles Bell (Committee Member) Subjects: Biochemistry; Biology; Microbiology; Molecular Biology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2015, Biological Sciences

RNA has emerged as a versatile and multi-faceted player in gene expression and the informational metabolism of living cells. RNA molecules can function by virtue of their sequences, storing or transmitting genetic information, as well as by forming complex three-dimensional (3D) structures that can bind specifically to proteins, small molecules or other RNA or DNA molecules to carry out diverse recognition functions, including chemical catalysis. In fact, large RNA-based molecular machines, like the ribosome and spliceosome, are now known to be RNA enzymes or “ribozymes” that rely on complex RNA 3D structure to sequentially bind and release their macro-molecular substrates and co-factors. As a result of revolutions in RNA 3D structure determination and high-throughput DNA and RNA sequencing, on-line databases are brimming with new structure and sequence data. The large amounts of new data are creating new challenges in data management, curation, search, visualization, and access. For example ribosomes have been solved in many different functional states, with tRNAs variously bound to the A-, P-, or E-sites, or associated with different translation factors (i.e. initiation, elongation, termination or recycling factors) or antibiotics. Bound tRNAs may be cognate, near cognate or non-cognate to the bound mRNA codon sequences present at the A-, P- or E-sites of the ribosome. Detailed and accurate functional annotations are needed to enable focused database searches for specific states and bound ligands and to uncover new relationships regarding structure, function, and evolution of RNA molecules and their complexes. As large numbers of new structures are accumulating in databases faster than they can be manually annotated, automated annotation procedures need to be developed and deployed by databases such as the Nucleic Acid Database (NDB). In addition to annotation of individual structures, related structures must be identified, compared and clustered, and representat (open full item for complete abstract)

Committee: Neocles Leontis Ph.D. (Advisor); R. Marshall Wilson Ph.D. (Other); Craig Zirbel Ph.D. (Committee Member); Carol Heckman Ph.D. (Committee Member); George Bullerjahn Ph.D. (Committee Member) Subjects: Biology

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)

Committee: Soumitra Basu (Advisor); Sanjaya Abeysirigunawardena (Committee Member); Hamza Balci (Committee Member); Jennifer McDonough (Committee Member); Yaorong Zheng (Committee Member) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2024, Microbiology

Assembly of ribosomes in bacteria involves the folding of three large ribosomal (r)RNAs and the binding of ~50 ribosomal proteins. In the test tube, active ribosomes can be reconstituted using just its constitutive components. These reconstitutions require non-physiological conditions. This shows that the information needed to form functional subunits is an intrinsic characteristic of these molecules. In the cell, ribosome assembly occurs co-transcriptionally and involves rRNA modification and rRNA processing. Cells employ diverse host of factors, known as assembly factors, that facilitate the assembly process and ensure the production of high-fidelity ribosomes. In most bacteria, rRNAs are transcribed from operons. In Escherichia coli, seven operons each encode the 16S rRNA, one or two tRNAs, 23S rRNA, and 5S rRNA. Upstream of the 16S rRNA are RNA elements (boxB, boxA, and boxC) that recruit proteins that form the rrn transcription antitermination complex, modifying RNA polymerase activity. Precursor RNA sequences, known as leader (5′) and trailer (3′) sequences, flank each gene. These precursor RNA segments must be removed to produce the mature length rRNA. For both the 16S and 23S rRNAs, flanking RNA sequences are complementary, forming long helices known as leader-trailer helices. In Chapter 2, I report the role the pre-16S rRNA has in the biogenesis of the 30S subunit. Using the orthogonal ribosome system, I measured the effect mutations to the pre-16S rRNA had on the translational activity of 30S subunits. I found that a leader-trailer helix of at least 15 base pairs is absolutely critical to forming functional subunits in the cell. Deletion of other elements, such as antitermination elements (boxA) and leader helices (hA and hB), of the pre-16S resulted in modest drops in activity. Interestingly, subunits formed in the absence of any of these elements of the pre-16S had defects in translational fidelity. This work shows the critical role of the leader-trai (open full item for complete abstract)

Committee: Kurt Fredrick (Advisor); Michael Kearse (Committee Member); Igor Jouline (Committee Member); Irina Artsimovitch (Committee Member) Subjects: Biochemistry; Biology; Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2024, Biochemistry Program, Ohio State

Canonical eukaryotic messenger RNA (mRNA) translation primarily uses the heterotrimeric eukaryotic initiation factor 2 (eIF2) to deliver initiator transfer RNA (tRNA) to the 40S ribosomal subunit. Additionally, at least three other factors have been shown to deliver initiator tRNA, namely eIF2A, eIF2D, and the MCT-1•DENR complex, to the 40S ribosomal subunit. eIF2A, a monomer non-homologous to eIF2, was the first factor identified to deliver the initiator tRNA in eukaryotes. Further investigation into the mechanism of eIF2A dwindled once eIF2 was found to be the primary initiator tRNA carrier. The current model in the field posits that eIF2A delivers initiator tRNA to specific transcripts with near-cognate non-AUG start codons during normal conditions or cell stress. Biochemical analyses to verify the function of eIF2A were lacking due to the inability to achieve a high yield of soluble recombinant protein. Therefore, the exact function and mechanism of eIF2A in translation remains largely enigmatic. eIF2A depletion does not impact global translation levels in yeast, mouse, or human cells as shown by radiolabeled amino acid incorporation, polysome profiling, and ribosome profiling approaches. While eIF2A does not function in a major initiation pathway, this factor has been implicated in tumor progression and proper lipid metabolism in mice, indicating the importance of eIF2A in some biological processes. Multiple reports suggest that eIF2A is non-cytosolic, which is uncharacteristic of an initiation factor. During normal physiological conditions, eIF2A has specifically been reported to localize to the nucleus, endoplasmic reticulum, or mitochondria depending on the cell type. How and why eIF2A is kept away from the translation initiation machinery is unknown, but it may be a mechanism for cells to only use eIF2A in specific conditions. To gain insight into the function of eIF2A, we first developed a robust method to recombinantly express and purify eIF2A that yielde (open full item for complete abstract)

Committee: Michael Kearse (Advisor); Karin Musier-Forsyth (Committee Member); Jane Jackman (Committee Member); Kurt Fredrick (Committee Member) Subjects: Biochemistry

PHD, Kent State University, 2023, College of Arts and Sciences / Department of Chemistry and Biochemistry

The ribosome is the ribonucleoprotein machine that carries out protein biosynthesis in all forms of life. Perfect synchronization between rRNA transcription, folding, post-transcriptional modification, maturation, and assembly of r-proteins is essential for the rapid formation of structurally and functionally accurate ribosomes. Many RNA nucleotide modification enzymes may function as assembly factors that oversee the accuracy of ribosome assembly. Ribosomal small subunit methyltransferase G (RsmG) is responsible for N7 methylation in G527 of 16S rRNA. In the Chapter 1 of my thesis work, the preferred rRNA substrate for RsmG binding and activity will be investigated. Various r-proteins that bind to the 5′-domain can influence RsmG binding. Hence, the effect of r-proteins on RsmG binding and activity will also be investigated. The data obtained will be used to identify the role of RsmG on rapidly generating functional ribosomes. Even though ribosomes are known to be synthesized with a unique rRNA and r-protein composition, various subpopulations of ribosomes has been discovered under various environmental stress conditions. These functionally specialized heterogeneous ribosomes are commonly known as “stress ribosomes” and results in the selective synthesis of about 10% of total cellular proteins. Ribosomal small subunit pseudouridine synthase A (RsuA) is found to be one of the survival proteins expressed in bacteria under stress. RsuA is responsible for the pseudouridylation of U516 in 16S rRNA. In the Chapter 2 of my thesis work, streptomycin stress-induced growth defects in a RsuA knock-out strain of E. coli will be investigated. The growth curves of wildtype and RsuA knock-out E. coli strains will be compared to demonstrate the influence of RsuA for bacterial growth under streptomycin stress. Wildtype and a catalytically inactive RsuA will be exogenously expressed in the RsuA knock-out strain to investigate the effect of the pseudouridylation activity of RsuA t (open full item for complete abstract)

Committee: Sanjaya Abeysirigunawardena (Advisor); Soumitra Basu (Committee Member); Robert Twieg (Committee Member); Hamza Balci (Committee Member); Manabu Kurokawa (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Chemistry

Honors Theses, Ohio Dominican University, 2023, Honors Theses

Antibiotic resistance is one of the world's fastest growing and most prevalent problems today. The influx of antibiotics within our environment from inadequate antibiotic stewardship has led to surge of drug resistant microorganisms. New drug development is imperative to combat infections caused by drug resistant pathogens. Bacterial translation, the process of protein synthesis by the ribosome, is a common target for new antibiotic development. Elongation factor P (EF-P) is a universally conserved protein that alleviates ribosomal pausing by aiding in peptide bonding at polyproline motifs. We characterize the role of EF-P in clinically related phenotypes within A. baylyi. EF-P is required for biofilm formation, surface associated motility, and resistance to beta-lactams and carbapenems within A. baylyi. The data we present holds hope for future drug development targeting EF-P in pathogens closely related to A. baylyi.

Committee: Anne Witzky (Advisor); Emily Post (Committee Member); Michael Doughtery (Committee Member) Subjects: Biology; Microbiology

Doctor of Philosophy, The Ohio State University, 2022, Biochemistry Program, Ohio State

The bacterial phylum, Bacteroidetes, is a remarkably diverse and ubiquitous lineage whose members are profoundly integral to the ecological niches they occupy such as the mammalian gut, the rhizosphere, and the ocean microbiome. They also possess a wealth of notable features that distinguishes them from other groups of bacteria. Despite their apparent ecological importance and the intriguing characteristics that they possess, they remain a poorly studied group. This deficiency in characterization is readily evident with regards to translation initiation in the Bacteroidetes, which is contrary to our common understanding of initiation in more well-studied bacteria. Among prokaryotic genomes, many protein coding genes have a Shine Dalgarno (SD) motif proximal to the start codon which serves to recruit ribosomes to that site. Translation initiation is often facilitated by pairing of the SD sequence to its motific counterpart, the anti-SD (ASD), on the 16S rRNA of the ribosome. This SD-ASD interaction was thought to be the predominant mechanism of translation initiation among bacteria and in many cases indispensable. Yet, SD sequences are greatly underrepresented in the genomes of Bacteroidetes, suggesting that the SD-ASD mechanism has become largely irrelevant. In the absence of SD sequences, alternative translational determinants must substitute and be more prominent in the Bacteroidetes. In this study, we identify translational determinants in the Bacteroidetes that supplant the SD motif. We observe similar alternative determinants in other organisms such as Escherichia coli that do use the SD sequences, but less so in organisms such as B. subtilis which relies heavily on SD sequences. However, what remains uncertain is why the Bacteroidetes lineage has largely dispensed with the SD sequence. Another intriguing aspect of the Bacteroidetes is that their ribosomes virtually retain the complete ASD sequence. The increasing irrelevance of the SD sequence should lessen t (open full item for complete abstract)

Committee: Kurt Fredrick (Advisor) Subjects: Biochemistry; Genetics; Microbiology; Molecular Biology

PHD, Kent State University, 2021, College of Arts and Sciences / Department of Chemistry and Biochemistry

Ribonucleotide modifications are ubiquitous in RNAs, including various types of non-coding RNAs and messenger RNA (mRNA). Pseudouridine (Ψ) is the most abundant single nucleotide modification in non-coding RNAs. With the advancement of high throughput sequencing, pseudouridine was recently found in mRNAs, and it is the second most abundant modified nucleotide in mRNAs. Besides its presence in various types of RNAs, the exact biological role of pseudouridine is not known. In general, the presence of pseudouridine increases RNA stability. In addition, mRNA pseudouridylation might play a role in epitranscriptomic gene expression regulation. Pseudouridine is an isoform of canonical uridine, and isomerization reaction is catalyzed by various pseudouridine synthases. Like other nucleotide modification enzymes, pseudouridine synthases also have important auxiliary functions besides their primary pseudouridylation activity. Ribosomal RNA (rRNA) from all three domains of life contains pseudouridines that clustered within or closer to functionally important regions of the ribosome. There are eleven pseudouridines in the bacterial ribosome. However, the bacterial small-subunit rRNA, 16S rRNA, contains only a single pseudouridine residue, Ψ516. Ribosomal RNA pseudouridine synthase A introduces the Ψ516 modification to the 16S helix 18. The 16S rRNA helix 18 forms a functional pseudoknot structure that is essential for ribosome activity. This dissertation work explores the preferred rRNA-protein complex for the binding and optimal activity of the pseudouridylation enzyme RsuA. Furthermore, the importance of protein RsuA and Ψ516 modification in developing the bacterial resistance towards streptomycin was also investigated. In addition, pseudouridine readers that interact with various pseudouridylated RNAs were also investigated. The various biophysical techniques and biological assays used in this research included Fοster Resonance Energy Transfer-(FRET), Pseudo-Seq, Ci (open full item for complete abstract)

Committee: Sanjaya Abeysirigunawardena (Advisor) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, Case Western Reserve University, 2021, Biology

Schistosome infection affects over 250 million people worldwide, leading to extensive morbidity and death. Although this parasitic helminth immiserates millions annually, significant gaps exist in our understanding of translational regulation. Further analysis of the basic biology of schistosomes is required to develop new, more effective treatment modalities. In this thesis, we perform global translational analysis of cercariae and early schistosomula and transcriptomic and proteomic analysis of cercariae. The cercariae are comprised of two macrostructures, the head and the tail. The cercarial head has limited motility but contains proteases and mucins required for host invasion. The cercarial tail is highly motile and is necessary for swimming and motility. Given the cercarial tail functions, we hypothesize that the tail requires translation to maintain metabolism for motility. Given that cercarial transformation is not affected by translational inhibition, we hypothesize that the cercarial head is translationally repressed. In this thesis, we present two major studies. The first study directly tested the global translation rates in cercarial heads and tails. We found that neither the head nor tail undergoes transcription, translation is also severely limited in the cercarial head, but to the contrary, the cercarial tail has extensive translational activity. We also found that translation is required for swimming behavior. The global translation analysis did not identify the mechanisms that control these differences in translation, so we performed transcriptomic and proteomic analyses of cercarial heads and tails. In the second study, we analyze heads' and tails' transcriptomes and proteomes. We found that the probable drivers of translational differences in heads and tails are the storage and ratios of ribosomal components. The pattern of increased ribosomal component proteins extends into schistosomula and paired adults as well. We also report that transcri (open full item for complete abstract)

Committee: Emmitt Jolly PhD (Advisor); Blanton Tolbert PhD (Committee Member); Brian McDermott PhD (Committee Member); Chris Cullis PhD (Committee Member); Yolanda Fortenberry PhD (Committee Chair) Subjects: Biology; Biomedical Research

PHD, Kent State University, 2021, College of Arts and Sciences / Department of Chemistry and Biochemistry

The ribosome is a ribonucleoprotein molecular machine that biosynthesizes proteins in all living cells. The biogenesis of ribosomes is crucial for the survival of all living organisms. In cells, ribosomes are assembled from ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) with the assistance of assembly factors such as helicases, modification enzymes, and RNases. These trans-acting factors play a vital role in synchronizing processes such as transcription, folding, modification, and maturation of rRNA, ribosome assembly, and quality control that cooccur during ribosome biogenesis. This dissertation work shows the ability of the rRNA nucleotide modification enzyme RsmC to influence ribosome assembly as a trans-acting factor during ribosome biogenesis. The protein RsmC was found to be functioning as an RNA chaperone and an RNA annealing protein in addition to its well-characterized methyltransferase activity. RsmC increases the rate of annealing of the two helix 34 (h34) RNA strands and denatures a non-native secondary structure in one of the strands (3h34). Mutations at the methylation site and mutations that influence the non-native secondary structure of RsmC substrate strand 3h34 weakens both the binding of RsmC to its substrate RNA and its chaperone and RNA strand annealing activity. The inability of RsmC to bind to mutant rRNA may slow down the 30S assembly and may also produce sub-optimally active ribosomes. Additionally, I have discovered a novel heptapeptide (3h34P1) that binds to the RsmC substrate RNA strand (3h34) using the phage display method during my doctoral work. This peptide binds tightly to its target RNA, alters the structure of the helix 34, and inhibits the RsmC binding. Furthermore, the 3h34P1 peptide showed bacterial growth defects that can be likely due to the inhibition of ribosome assembly. This peptide could be developed into a potent antibacterial agent against pathogenic bacteria in the future by chemically modifying the peptide (open full item for complete abstract)

Committee: Sanjaya Abeysirigunawardena PhD (Advisor) Subjects: Biochemistry; Chemistry

BS, Kent State University, 2021, College of Arts and Sciences / Department of Chemistry and Biochemistry

Ribosome is a ribonucleoprotein complex composed of three ribosomal RNAs and approximately 80 ribosomal proteins. It catalyzes the synthesis of proteins that regulate various biological processes in an organism, so proper ribosomal assembly is essential. In-vivo ribosomal biogenesis integrates number of processes, such as rRNA transcription, ribosome maturation, ribosome assembly, quality control, etc. It is speculated that r-proteins bind to rRNA co-transcriptionally along with other processes, such as nucleotide modification, rRNA folding, etc. Various rRNA modification enzymes also participate in ribosome biogenesis as assembly factor. More than 100 nucleotide modifications are distributed in various classes of RNAs in all kingdoms of life, but their functional role has not been discovered yet. In bacteria, 10 enzymes control 11 nucleotide modifications in 16s rRNA. Lack of those rRNA modifications result in growth defects, disease, and disorders by influencing ribosome assembly, but the direct link between nucleotide modification and ribosome assembly defects has not been determined yet. Ribosomal small subunit methyltransferase C (RsmC), a ribosomal RNA modification enzyme, catalyzes the transferring of methyl group from S-adenosyl methionine (SAM) to exocyclic amine of G1207 of 16S helix 34. It has two tandemly duplicated domain, methyltransferase active C-terminal domain and catalytically inert N-terminal domain. This project is focused on identifying the RNA strand that specifically binds to the N-terminal domain and also to evaluate the role of the RsmC N-terminal domain in the RNA chaperon activity. Various 16S helix 34 mutants are used to elucidate the biological role of RsmC under various conditions that generate ribosome mutations.

Committee: Sanjaya Abeysirigunawardena Ph.D. (Advisor); Yaorong Zheng Ph.D. (Committee Member); Xiaozhen Mou Ph.D. (Committee Member); Elda Hegmann Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2021, Biochemistry

Reprogramming of mRNA translation is crucial for coping with diverse stressors. In this thesis, using ribosome footprinting, I characterized how the translatome of mammalian cells is remodeled in response to the increasing intensities of hypertonic stress. My work revealed how different stress intensities can alter the cellular strategy to cope with stress. While mild stress promotes the translation of various chaperones and osmotic stress-specific transcription factors, severe stress dramatically inhibits it. Nevertheless, even under severe stress conditions, cells can initiate translation on the specific mRNAs coding for ribosomal and nuclear-encoded mitochondrial proteins. However, 80S ribosomes were unable to elongate and were stalled at the start codon as a fail-safe mechanism to ensure no protein is made under these conditions. Ribosomal pausing correlated with the higher residence of eIF5B on 80S ribosomes. Stalling was reversed upon the exposure of stressed cells to isotonic media and key proteins of the Integrated Stress Response (ISR) were expressed to promote recovery. My data suggest that hypertonic stress not only inhibits the recruitment of ribosomes to the mRNAs but also prevents the already assembled 80S ribosome to transition into productive elongation. My work revealed other peculiar features specific to severe hypertonic stress : pervasive leaky scanning and the accumulation of non-classical, shorter (21-22 nt) ribosome footprints, corresponding to different conformational states of ribosomes. I also examined how the translatome is reshaped as cells adapt to mild hypertonicity in human corneal cells, a cellular model to study the pathophysiology of dry eye syndrome. This osmoadaptive state depended on the coordinated actions of the plasma membrane amino acid transporter, SNAT2, and mTORC1. SNAT2 functioned as a switch that turned on the translation of specific mRNAs to help with the survival under these conditions. My work reveale (open full item for complete abstract)

Committee: Maria Hatzoglou (Advisor); William Merrick (Committee Chair); Kristian Baker (Committee Member); Eckhard Jankowsky (Committee Member); Donny Licatalosi (Committee Member); Martin Snider (Committee Member) Subjects: Biochemistry

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

Short Interspersed Nuclear Elements (SINEs) are non-autonomous mobile elements present in eukaryotic genomes. SINEs are mobilized in the form of a transcribed RNA intermediate in a `copy and paste' process referred to as retrotransposition, by making use of the enzymes encoded by an autonomous element pair, or a Long Interspersed Nuclear Element (LINE). In turn, the active LINE is able to mobilize its own mRNA (termed retrotransposition in cis) or a SINE RNA (in trans). SINE insertions have been linked to not just genetic diversity but also naturally and artificially selected phenotypes in humans and non-human mammals. Within these host genomes, SINEs differ in their activities and evolutionary histories. Unlike a LINE, which contains an RNA II polymerase promoter, a SINE is known for its internal polymerase III promoter which has contributed to its structure and an advantage in utilizing the mobile proteins from LINE, for example in the human Alu by attaching onto the ribosome via conserved structure involving signal recognition particles (SRP). The active and most predominant SINE in human genomes, Alu, is derived from 7SL RNA; the active SINE in canine genomes, SINE_Cf is derived from a tRNALys, and active SINEs in murine genomes, B1 and B2, are derived from either 7SL and tRNA, respectively, each of the above in an evolutionary independent manner. Members of the currently active Alu/LINE pair, of the specific AluY (i.e., for `young') and L1Hs (`L1' for `active' and “Hs' for `human specific') have been very well studied and their mobilization detailed in an elegant model. Alu RNA adopts the conserved structure and cellular binding partners as it's ancestral 7SL RNA molecule, SRP9/14, which directs the Alu intermediate to the ribosome. In the currently understood Alu/L1Hs model, while docked on the ribosome, the Alu RNA intermediate `piggybacks' the translated L1Hs ORF2p at the ribosome, which functions to transport the intermediate to the nucleus and reverse tra (open full item for complete abstract)

Committee: Julia Halo Ph. D. (Advisor); Raymond Larsen Ph. D. (Committee Member); Vipaporn Phuntumart Ph. D. (Committee Member) Subjects: Biology; Genetics; Molecular Biology

BS, Kent State University, 2019, College of Arts and Sciences / Department of Chemistry and Biochemistry

Ribosomes are the molecular machines that carry out protein biosynthesis in all living organisms. They are composed of three different ribosomal RNAs and more than 50 ribosomal proteins. The thermodynamics and kinetics of in vitro ribosomal assembly have been studied extensively. However, during ribosome biogenesis, in vivo ribosome assembly occurs concurrently with transcription, folding, post-transcriptional modifications, and processing of rRNA. Unfortunately, the effects post-transcriptional RNA modification enzymes on ribosomal assembly are understudied. My project in Abey lab is to investigate how RNA modification enzyme RsmG influences 30S bacterial ribosome assembly. I have developed a FRET based assay to monitor binding of RsmG to ribosomal RNA, which will allow us to determine the binding affinity of RsmG to RNA and thus calculate thermodynamic cooperativity between RsmG enzyme and ribosomal proteins. Our findings give us more insight to how modification enzymes modulate the hierarchy of protein addition during ribosome biogenesis.

Committee: Sanjaya Abeysirigunawardena PhD (Advisor); Gail Fraizer PhD, MPH (Committee Member); Hanbin Mao PhD (Committee Member); Alexander Seed PhD (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2018, Pathology

Hereditary cancer syndromes serve as powerful models for uncovering cancer-relevant genes. Identification of cancer-predisposing genes not only facilitates gene-enabled molecular diagnosis, cancer risk assessment and gene-specific clinical management, but also offers valuable insights into therapeutically targetable pathways contributing to sporadic tumorigenesis. We study Cowden syndrome (CS) as a model for germline predisposition to cancer. CS is an autosomal dominant disorder characterized by high lifetime risks of epithelial cancers, with ~50% of patients wildtype for known predisposition genes. Using whole-exome and Sanger sequencing of a multi-generation CS family with thyroid and other cancers, we identified a pathogenic missense heterozygous variant in SEC23B (c.1781T>G, p.Val594Gly) that segregated with phenotype. SEC23B encodes Sec23 Homolog B, a component of coat protein complex II (COPII), transporting proteins from endoplasmic reticulum (ER) to Golgi apparatus. Interestingly, germline homozygous or compound heterozygous SEC23B mutations cause an unrelated disorder, Congenital Dyserythropoietic Anemia Type II, and SEC23B-deficient mice show secretory organ degeneration due to ER stress-associated apoptosis. Extending our genetic studies, we also found germline heterozygous SEC23B variants in 3/96 (3%) unrelated mutation negative CS probands with thyroid cancer, and in The Cancer Genome Atlas (TCGA), representing apparently sporadic cancers. We note that TCGA thyroid cancer dataset is enriched for unique germline deleterious SEC23B variants that were associated with a significantly younger age at onset. At the functional level, by characterizing the p.Val594Gly variant in a non-malignant thyroid cell line, we provide evidence that it is a functional mutation that results in ER stress-mediated cell colony formation and survival, growth, and invasion, reflecting aspects of a cancer phenotype. Importantly, these phenotypes occurred with absence of CDA II- (open full item for complete abstract)

Committee: Charis Eng MD, PhD (Advisor); Shigemi Matsuyama PhD (Committee Chair); Clive Hamlin PhD (Committee Member); George Stark PhD (Committee Member); Ruth Keri PhD (Committee Member); Thomas LaFramboise PhD (Committee Member) Subjects: Bioinformatics; Biomedical Research; Cellular Biology; Genetics

Doctor of Philosophy, The Ohio State University, 2016, Microbiology

In cells, ribosomes translate the genetic information in messenger RNA to synthesize proteins. Aminoglycosides (AGs) represent a very large group of antimicrobials that target this crucial process of translation in bacteria. They target helix h44 of 16S rRNA and H69 of 23S rRNA, and cause miscoding, inhibit mRNA-tRNA translocation and inhibit ribosome recycling. The role of each of the two AG binding sites in inhibition of translocation has remained unclear. Binding of AGs to h44 is believed to stabilize A site tRNA and cause miscoding and inhibition of translocation. Binding of AG to H69 is suggested to inhibit ribosome recycling. In Chapter 2 I analyzed the role of the two AG binding sites to inhibition of mRNA-tRNA translocation. My data suggest that h44 site is central to the activity of translocation inhibition of NEO and TOB, representatives of 4,5- and 4,6-substituted 2-deoxystrptamine AGs. By contrast, the H69 site plays no role in translocation inhibition by TOB and only a minor role in translocation inhibition by NEO. This work sheds light on the roles of the two AG binding sites in AG activity in ribosomes. With the emergence of AG-resistant bacterial strains, the need to develop novel AGs with improved bacterial ribosome-targeting capability and enhanced antimicrobial activity is more and more pressing. In Chapter 3 of this work, I evaluated 37 novel tobramycin (TOB) derivatives with various thioether substituents at the 6''-position. Thirteen of these displayed better antibacterial activity than the parent TOB while retaining ribosome-targeting specificity. Analysis of these compounds in vitro revealed three with clearly enhanced ribosome-targeting activity. These compounds are also poorer substrates of AG-modifying enzymes compared to TOB. This study identified promising antibiotic candidates and a possible general strategy to increase the efficacy of ribosome-targeting antibiotics. Chapter 4 describes my discovery that eleven AG derivatives can inh (open full item for complete abstract)

Committee: Kurt Fredrick (Advisor); Tina Henkin (Committee Member); Mike Ibba (Committee Member); Jane Jackman (Committee Member) Subjects: Microbiology

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

Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the efp deficient strain, with up to a 45-fold increase in ribosome density observed at the pausing site. The unexpectedly low fraction of PPX motifs that produce a pause in translation led us to investigate the possible role of sequences upstream of PPX. Our data indicate that EF-P dependent pauses are strongly affected by sequences upstream of the PPX pattern. We found that residues as far as 3 codons upstream of the ribosomal peptidyl-tRNA site had a dramatic effect on whether or not a particular PPX motif triggered a ribosomal pause, while internal Shine Dalgarno sequences upstream of the motif had no effect on EF-P dependent translation efficiency. Increased ribosome occupancy at particular stall sites did not reliably correlate with a decrease in total protein levels, suggesting that in many cases other factors compensate for the potentially deleterious effects of stalling on protein synthesis. These findings indicate that the ability of a given PPX motif to initiate an EF-P-alleviated stall is strongly influenced by its local context, and that other indirect post-transcriptional effects determine the influence of such stalls on protein levels within the cell.

Committee: Michael Ibba Dr. (Advisor); Irina Artsimovitch Dr. (Committee Member); Kurt Fredrick Dr. (Committee Member); Jane Jackman Dr. (Committee Member) Subjects: Biochemistry; Genetics; Microbiology