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A significant number of known human genetic diseases is associated with nonsense mutations leading to the introduction of a premature termination codon into the coding sequence. A termination codon can be read through by its near-cognate tRNA (tRNA with two anticodon nucleotides base-pairing with a stop codon); potentially generating C-terminally extended protein variants. In yeast, UGA stop codon was described to be read through by tRNA-Trp and tRNA-Cys. Similar was observed for tRNA-Trp in human HEK293T cell line. The aim of this thesis was to investigate if human tRNA-Cys can act as a near-cognate tRNA in human HEK293T cell line. There are two isoacceptors which constitute the tRNA-Cys family, with ACA and GCA anticodon. There are 1 and 23 isodecoders to the ACA and GCA anticodons, respectively. Here, altogether as many as nine tRNA-Cys isodecoders (distinct in their sequence and with varying levels of expression) were tested for their ability to increase UGA readthrough in HEK293T using p2luci and pSGDluc dual-luciferase reporter vectors. In both p2luci and pSGDluc, we observed that at least one tRNA-Cys isodecoder, tRNA-Cys-GCA-4-1, is capable of significantly elevating the UGA readthrough levels when overexpressed in HEK293T. This indicates that similarly to yeast, tRNA-Cys is capable of...

Non-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for gene regulation and other functional roles and have also found to contribute to pathogenesis in humans. The increased size and structural complexity require novel tools to study their interactions beyond RNA interference. Synthetic antibodies are classic tools and therapeutics utilized to study and treat proteins involved in human disease. Likewise we hypothesize that structured RNAs can also take advantage of synthetic antibodies to probe their functions and be utilized as therapeutics. Currently, antibodies have been raised against microbial riboswitches and other structured RNAs of single-celled organisms, and only one human structured RNA to the best of our knowledge. However, no one has yet to create a synthetic antibody capable of behaving as a therapeutic against a structured RNA. We therefore sought to raise an antibody Fab against a structured RNA, human initiator tRNA, a model oncogenic non-coding RNA and demonstrate its efficacy in vitro. We then characterized the antibody and explored delivery options in cancer cells including the use of nanoparticle delivery systems. With the emerging transcriptome revealing new ncRNAs implicated in human disease, our research has begun to address a new therapeutic strategy, laying down the foundation for the future of structured RNA-targeted therapies.

Saccharomyces cerevisiae are unicellular organisms that are highly adaptable to acute changes in nutrient availability. The two main signaling pathways that allow S. cerevisiae to sense and respond to changes in glucose availability in the environment are the conserved cAMP/PKA and AMPK/Snf1 kinase-dependent pathways. The conserved TORC1 pathway is primarily responsible for allowing cells to respond to the availability of nitrogen. Studies have shown that S. cerevisiae, but not mammalian and plant cells, regulate nuclear tRNA trafficking in response to nutrient stress. Here, we show that the yeast species of the Saccharomyces genus, but not Schizosaccharomyces pombe and Kluyveromyces lactis specifically regulate nuclear tRNA export in response to nutrient stress, providing further evidence that regulation of nuclear tRNA export in response to nutrient availability is not evolutionarily conserved. We also established that amino acid and nitrogen starvation affects nuclear export of a subset of tRNAs in S. cerevisiae. Inhibition of TORC1 signaling by rapamycin treatment, which simulates nitrogen starvation, also affects nuclear export of the same subset of tRNAs, suggesting that the TORC1 signaling pathway plays a role in regulating nuclear export of the tRNAs in response to nitrogen level. Regulation of nuclear export of these tRNAs by nitrogen deprivation is most likely due to an effect on the function of the nuclear tRNA export receptors, as overexpression of the tRNA export receptor, Los1p, restores export of the tRNAs during nitrogen starvation. These findings suggest that the TORC1 signaling pathway may, in part, regulate nuclear export of the tRNAs by affecting the function of the tRNA export receptors. In contrast to amino acid and nitrogen starvation, glucose depletion affects nuclear export of all tRNA species in S. cerevisiae. Evidence obtained suggests that nuclear retention of tRNA in cells deprived of glucose is due to a block in nuclear re-import of the nuclear tRNA export receptors. Retention of the receptors in the cytoplasm is not caused by activation of Snf1p, but by the inactivation of PKA during glucose deprivation. Furthermore, regulation of nuclear re-import of the receptors is not due to phosphorylation of the tRNA export receptors by PKA. However, PKA phosphorylates known components of the tRNA export machinery. A model that is consistent with the data is that PKA and an unknown mechanism regulate the activity of these components or an unidentified protein(s) to control nuclear re-import of the receptors in response to glucose availability.

Despite the high mortality rate of ovarian cancer, there are few selective biomarkers that detect its progression and none have become successful targets for therapy. A complex microenvironment that promotes angiogenesis, reduces immune responses and alters the integrity of the surrounding matrix is involved through the biology of ovarian cancer. Previous studies done by our lab and collaborators indicated that extracellular threonyl-tRNA synthetase (TARS) is a pro-angiogenic mediator of the ovarian tumor microenvironment, which is secreted in response to inflammatory signals, and actively promotes angiogenesis. In order to better understand the mechanisms underlying the angiogenic effects of TARS in ovarian cancer, it is essential to identify whether it directly affects ovarian tumor growth and invasion. Preliminary evidence indicated that TARS is secreted from ovarian cancer cells in response to TNF-α and TARS exhibits extracellular angiogenic activity. In previous studies, TARS was shown to significantly increase migration of HUVECs in a transwell assay to an extent that was similar to VEGF. The purpose of this project was to establish a role for TARS in tumor progression and its potential as a diagnostic marker using an animal model of ovarian cancer. The hypothesis tested is that TARS plays a key role in the angiogenic and invasive potential of ovarian cancer, and TARS inhibition will reduce the angiogenic effect of tumor cells which is reflected by measurement of intratumor microvessel density (MVD). The study tested the effect of BC194-mediated TARS inhibition on the development of ovarian tumors in ID8 mouse model. We found a positive correlation between TARS expression and ovarian cancer progression, and TARS inhibition with BC194 reduce the progression of ovarian cancer. These data suggest that TARS has an important role in the tumor microenvironment and that TARS inhibition should be further investigated as a therapy for ovarian and other angiogenic cancers.

The aminoacyl-tRNA synthetases are among the major protein components in the translation machinery. These essential proteins are responsible for charging their cognate tRNAs with the correct amino acid. Mammalian arginyl-tRNA synthetase (ArgRS), unlike all other eukaryotic aminoacyl-tRNA synthetases, is unique due to the coexistence of two structurally distinct forms of the same enzyme within the same cell: a complexed (or high molecular weight) form that is part of the multi-synthetase complex, and a free (or low molecular weight) form. Until now, not much information is known as to why the cell would synthesize and utilize two different forms of the same enzyme. Do the two forms of ArgRS perform similar or different biological functions? The main hypothesis that was originally proposed is that only the complexed form of ArgRS plays a crucial role in protein synthesis, while the free form of this enzyme participates in the ubiquitination pathway by tagging proteins with acidic NH2-termini (destined for degradation) with an arginine residue on their NH2-terminal end which will serve as a signal for ubiquitin-mediated destruction. Based on my studies, the data indicate that the high molecular weight form of ArgRS, which is present exclusively as an integral component of the multisynthetase complex, is essential for normal protein synthesis and growth of CHO cells even when low molecular weight, free ArgRS is present and Arg-tRNA continues to be synthesized at close to wild type levels. Based on these observations, we can conclude that Arg-tRNA generated by the synthetase complex is a more efficient precursor for protein synthesis than Arg-tRNA generated by free ArgRS, exactly as would be predicted by the channeling model for mammalian translation. No phenotype has been determined for cells expressing only the complexed form of ArgRS, and no direct interaction has been observed between ArgRS and arginyl-tRNA-protein transferase (ATE). Based on this information, we suggest that the function(s) of the free form of ArgRS is either not necessary or is performed by the complexed form when the free form is missing.

Abstract “Biomimetic Aminoacylation” Svetlana K. Tzvetkova Doctor of Philosophy, 2008 Graduate Department of Chemistry University of Toronto The accuracy of ribosomal protein synthesis depends on the fidelity of highly specific enzymes, aminoacyl tRNA synthetases, towards amino acid – tRNA pairs. These biological catalysts are responsible for activating the amino acids as aminoacyl adenylates and for their subsequent attachment to the 2’- or 3’-OH at the 3’-terminal of the correct tRNA to give aminoacyl-tRNA. Extended diversity in protein structure and function could be achieved if non-natural side chains can be introduced in protein synthesis. This requires that the acceptor stem of a tRNA molecule be synthetically aminoacylated. The most widely used methods for charging tRNA with non-natural amino acids involve multi-step synthesis of an aminoacyl-pCpA and its consequent enzymatic ligation to truncated tRNA. No direct route to these species has been reported. We have developed a method for direct biomimetic aminoacylation of the 3’-terminal hydroxyls of tRNA. Our approach shows to be promising in reactions leading to direct 2’- or 3’-O-aminoacylation of not only nucleosides and nucleotides but also RNA in general and tRNA in particular. The system we have developed provides: 1) efficient activation of the amino acids as aminoacyl phosphates, analogues of the enzymatic intermediates, and 2) specific recognition of the 3’-terminal of tRNA by lanthanide ions present in the reaction. The aminoacylating reagents used in our studies were carefully selected to provide handles to follow the reaction: UV absorbance, fluorescence spectroscopy and 19F NMR. Lanthanide (III) ions can play a role similar to a key part of the aminoacyl tRNA synthetases – they bring the aminoacyl close to the 3’-terminal of tRNA, in this case by forming a bis-bidentate complex with the aminoacyl phosphate and the 2’,3’-diol functionality of the 3’-terminal adenosine. This process relies on the specificity towards the unique 3’-terminal diol on tRNA, provided by the metal ion and the simultaneous complexation of the aminoacyl phosphate.

Abstract “Biomimetic Aminoacylation” Svetlana K. Tzvetkova Doctor of Philosophy, 2008 Graduate Department of Chemistry University of Toronto The accuracy of ribosomal protein synthesis depends on the fidelity of highly specific enzymes, aminoacyl tRNA synthetases, towards amino acid – tRNA pairs. These biological catalysts are responsible for activating the amino acids as aminoacyl adenylates and for their subsequent attachment to the 2’- or 3’-OH at the 3’-terminal of the correct tRNA to give aminoacyl-tRNA. Extended diversity in protein structure and function could be achieved if non-natural side chains can be introduced in protein synthesis. This requires that the acceptor stem of a tRNA molecule be synthetically aminoacylated. The most widely used methods for charging tRNA with non-natural amino acids involve multi-step synthesis of an aminoacyl-pCpA and its consequent enzymatic ligation to truncated tRNA. No direct route to these species has been reported. We have developed a method for direct biomimetic aminoacylation of the 3’-terminal hydroxyls of tRNA. Our approach shows to be promising in reactions leading to direct 2’- or 3’-O-aminoacylation of not only nucleosides and nucleotides but also RNA in general and tRNA in particular. The system we have developed provides: 1) efficient activation of the amino acids as aminoacyl phosphates, analogues of the enzymatic intermediates, and 2) specific recognition of the 3’-terminal of tRNA by lanthanide ions present in the reaction. The aminoacylating reagents used in our studies were carefully selected to provide handles to follow the reaction: UV absorbance, fluorescence spectroscopy and 19F NMR. Lanthanide (III) ions can play a role similar to a key part of the aminoacyl tRNA synthetases – they bring the aminoacyl close to the 3’-terminal of tRNA, in this case by forming a bis-bidentate complex with the aminoacyl phosphate and the 2’,3’-diol functionality of the 3’-terminal adenosine. This process relies on the specificity towards the unique 3’-terminal diol on tRNA, provided by the metal ion and the simultaneous complexation of the aminoacyl phosphate.

This dissertation has two major sections. In Chapter II, complete mitochondrial (mt DNA) genome sequences were used to construct a hypothesis for affinities of most major lineages of rodents that arose quickly in the Eocene and were well established by the end of the Oligocene. Determining the relationships among extant members of such old lineages can be difficult. Two traditional schemes on subordinal classification of rodents have persisted for over a century, dividing rodents into either two or three suborders, with relationships among families or superfamilies remaining problematic. The mtDNA sequences for four new rodent taxa (Aplodontia, Cratogeomys, Erethizon, and Hystrix), along with previously published Euarchontoglires taxa, were analyzed under parsimony, likelihood, and Bayesian criteria. Likelihood and Bayesian analyses of the protein-coding genes converged on a single topology that weakly supported rodent monophyly and was significantly better than the parsimony trees. Analysis of the tRNAs failed to recover a monophyletic Rodentia and did not reach convergence on a stationary distribution after fifty million generations. Most relationships hypothesized in the likelihood topology have support from previous data. Mt tRNAs have been largely ignored with respect to molecular evolution or phylogenetic utility. In Chapter III, the mt tRNAs from 141 mammals were used to refine secondary structure models and examine their molecular evolution. Both H- and L-encoded tRNAs are AT-rich with different %G and GC-skew and a difference in skew between H- and L-strand stems. Proportion of W-C pairs is higher in the H-strand and GU/UG pairs are higher in the L-strand, suggesting increased mismatch compensation in L-strand tRNAs. Among rodents, the number of variable stem base-pairs was nearly 75% of that observed across all mammals combined. Compensatory base changes were present only at divergences of 4% or greater. Neither loop reduction nor an accumulation of deleterious mutations, both suggestive of mutational meltdown (Muller's ratchet), was observed. Mutations associated with human pathologies are correlated only with the coding strand, with H-strand tRNAs being linked to substantially more of these mutations.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 178-184).