For nearly five decades, the simple eukaryote Saccharomyces cerevisiae has been used as a model for understanding the eukaryotic cell cycle. One vein of this research has focused on understanding how chromosome structure is regulated in relation to the cell cycle. This work characterizes a new mechanism that modulates the chromatin organizing condensin complex, in hopes of furthering the understanding of chromosome structure regulation in eukaryotes. During mitosis, chromosomes are condensed to facilitate their segregation through a process mediated by the condensin complex. Upon interphase onset, condensation is reversed, allowing for efficient transcription and replication of chromosomes. This work demonstrates that Ycg1, the Cap-G subunit of budding yeast condensin, is cell-cycle regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. The cyclical expression of Ycg1 is unique amongst condensin subunits, and is established by a combination of cell cycle-regulated transcription and constitutive proteasomal degradation. Interestingly, when cyclical expression of Ycg1 is disrupted, condensin formation and chromosome association increases, and cells exhibit a delay in cell-cycle entry. These results demonstrate that Ycg1 levels limit condensin function, and suggest that regulating the expression of an individual condensin subunit helps to coordinate chromosome conformation with the cell cycle. These data, along with recent corroborating results in Drosophila melanogaster suggest that condensin regulation through limiting the expression of a single condensin subunit may be broadly conserved amongst eukaryotes.

In eukaryotes, sequences that code for the amino acid structure of proteins represent a small fraction of the total sequence space in the genome. These are referred to as coding sequences, whereas the remaining majority of the genome is designated as noncoding. Studies of translation, the process in which a ribosome decodes a coding sequence to synthesize proteins, have primarily focused on coding sequences, mainly due to the belief that translation outside of canonical coding sequences occurs rarely and with little impact on a cell. However, recently developed techniques such as ribosome profiling have revealed pervasive translation in a diverse set of noncoding sequences, including long noncoding RNAs (lncRNAs), introns, and both the 5’ and 3’ UTRs of mRNAs. Although proteins with amino acid sequences derived partially or entirely from noncoding regions may be functional, they will often be nonfunctional or toxic to the cell and therefore need to be removed. Translation outside of canonical coding regions may further expose the noncoding genome to selective pressure at the protein level, leading to the generation of novel functional proteins over evolutionary timescales. Despite the potentially significant impact of these processes on the cell, the cellular mechanisms that function to detect and triage translation in diverse noncoding regions, as well as how peptides that escape triage may evolve into novel functional proteins, remain poorly understood.This thesis will describe novel findings that offer new insight into the process of noncoding translation mitigation revealed by a combination of high-throughput systems-based approaches and validated by biochemical and genetic approaches. Chapter 1 will discuss general concepts in the translation of noncoding sequences and the relevant cellular systems and impacts on human health. Chapter 2 will discuss the results of a high-throughput reporter assay investigating translation in thousands of noncoding sequences from diverse sources. The results discussed in this chapter revealed two factors involved in the mitigation of proteins derived from noncoding sequences: C-terminal hydrophobicity and proteasomal degradation. Chapter 3 will build on Chapter 2 and discuss the results of a genome-wide CRISPR/Cas9 knockout screen that identified the BAG6/TRC35/RNF126 membrane protein chaperone complex as a key cellular pathway in the detection and degradation of proteins with translated noncoding sequences. Having identified the BAG6 complex as targeting a specific reporter of translation of the 3’ UTR in the AMD1 gene, a series of knockout cell lines validated these results and demonstrated the participation of two additional genes, SGTA and UBL4A. Through coimmunoprecipitation western blots and rescue assays with flow cytometry as a readout, we confirmed physical interaction between BAG6 and the 3’ UTR of AMD1, and a similar experiment confirmed interaction between BAG6 and a readthrough mutant of the SMAD4 tumor suppressor gene. Finally, by combining our high-throughput reporter library with our BAG6 knockout cell line, we demonstrated that BAG6 targets hydrophobic C-terminal tails in many noncoding sequences of diverse origin. Finally, Chapter 4 will discuss the evolutionary perspective of noncoding translation through analyses of the sequence content of human and mouse genomes. The findings of this chapter demonstrate a significant trend for increased uracil content in noncoding regions of the genome, which frequently results in the translation of hydrophobic amino acids. We also find that many functional translated noncoding peptides localize to membranes, providing a theoretical link between the shuttling of translated noncoding sequences to a protein complex involved in membrane protein quality control and the emergence of newly evolving proteins from the noncoding genome.

Termination of translation in eukaryotes occurs when a translating ribosome encounters a stop codon (UAA, UAG, or UGA) in its A site. This triggers the recruitment of translation termination factors eRF1, a tRNA-mimicking protein responsible for decoding the stop codon and catalyzing peptide release, and eRF3, a translational GTPase that stimulates peptide release in a GTP-hydrolysis-dependent manner. Upon successful stop codon decoding by eRF1, eRF3 carries out GTP hydrolysis and dissociates from the ribosome. eRF1 subsequently gets accommodated into the peptidyl transferase center (PTC) and catalyzes the release of the nascent peptide. The structures for the pre-accommodated eRF1 with eRF3 trapped on ribosome using non-hydrolysable GTP analogs as well as for the PTC-accommodated eRF1 have been solved using cryogenic electron-microscopy (cryo-EM). The structures reveal the binding mode and interactions between the release factors and the pre-termination complex. However, the mechanism of eRF3 GTPase activation and subsequent eRF1 accommodation into the PTC remains poorly understood. To address this knowledge gap, we used single-particle time-resolved cryo-EM (TRCEM) to capture the structures of intermediates formed during the termination process. For our TRCEM experiments, we first developed a Polydimethylsiloxane (PDMS)-based modular microfluidic mixing-spraying device with a SiO₂ internal coating. The device has a SiO₂-coated PDMS-based 3D splitting-and-recombination (SAR) micro-mixer capable of mixing two fluids within 0.5 ms with more than 90% efficiency. The SiO₂coating strengthens the PDMS channels and acts as a hydrophilic barrier preventing sample adsorption to the PDMS surface. The micro-mixer is connected to a glass microcapillary that acts as the reaction channel. Channels of different lengths can be used to vary the overall reaction time between 10 ms and 1000 ms. The microcapillary is connected to a 3D micro-sprayer for generating a 3D plume of sprayed droplets. A cryo-EM grid is passed through the spray cone to collect droplets and is rapidly plunged into liquid cryogen for vitrification. By using TRCEM as well as the conventional blotting method for cryo-EM sample preparation, we captured the reaction between a pre-termination (pre-TC) mimic and the ternary complex of eRF1, eRF3, and GTP at reaction times of 450 ms, 900 ms, 15 s, and 10 min. Classification of the cryo-EM data resulted in maps for five distinct factor-bound classes. Four maps belonged to intermediates with densities for eRF1 and eRF3 bound to the pre-TC in varying conformations. The fifth map had a density matching the PTC-accommodated eRF1. Population analysis allowed us to arrange the classes chronologically and track the events leading to GTPase activation during the termination process. Atomic model building and refinement allowed us to determine the hydrolysis state of the eRF3-bound GTP and revealed the catalytic mechanism for GTP-hydrolysis. The models revealed a potential mechanism for GDP dissociation post-GTP-hydrolysis.

This dissertation focuses on pre-mRNA 3'-end processing in eukaryotes, a crucial step in defining the 3'end of most protein-coding mRNAs. In vertebrates, two distinct molecular machines are involved: the canonical machinery, consisting of a Cleavage Factor (CF) module, Polyadenylation Specificity (PSF) module, Cleavage Stimulation Factor, and other complexes, and the U7 snRNP machinery (U7 machinery), which consist of a core U7 snRNP complex and the Histone Cleavage Complex (HCC). U7 snRNP is involved in replication-dependent histone pre-mRNA 3'-end processing. Interestingly, the cleavage modules of the canonical and U7 machinery share an endonuclease, CPSF73, that catalyzes the cleavage reaction for 3’-end processing of pre-mRNAs. CPSF73 also possesses 5’-3’ exonuclease activity in the U7 machinery. CPSF73 has been identified as a potential target for anticancer and antimalarial small-molecule inhibitors. Traditionally, CPSF73 nuclease activity has been demonstrated using a gel-based end-point assay, using radio-labeled or fluorescently labeled RNA substrates. In Chapter Two (Ch. 2) of this dissertation introduces a novel, real-time fluorescence assay to investigate CPSF73 nuclease activity. This efficient and high-throughput assay holds potential for identifying new CPSF73 inhibitors. Chapter Three (Ch. 3) of this dissertation delves into the structural characterization of the mammalian PSF (mPSF) module in complex with the second most frequent PAS variants, AUUAAA. Structure studies have revealed the molecular mechanism underlying mPSF recognition of the most common PAS sequence, AAUAAA. This study presents a cryo-EM structure of mPSF in complex with AUUAAA. While the binding modes remain highly similar between the two PAS variants, we observed conformational differences in the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 of AAUAAA. Furthermore, CPSF30 displayed conformational changes near the U2 nucleotide of AUUAAA. Attempts to explore the binding modes of two rare PAS sequences, AAGAAA and GAUAAA, were inconclusive due to a lack of RNA density in the EM maps. An atomic model of the ternary structure (CPSF160, WDR33, CPSF30) was produced using the EM map of the AAGAAA sample. The ternary structure revealed PAS recognizing residues to be disordered in CPSF30 (ZF2 and ZF3) and WDR33. Overall, this dissertation provides insights into the intricate mechanisms of pre-mRNA 3'-end processing in mammals, laying the groundwork for future studies and potentially leading to the development of novel inhibitors targeting CPSF73.

The mRNA coding for the ammonium inducible NADP-specific glutamate dehydrogenase (NADP-GDH) from Chlorella was studied in induced and uninduced cells to determine the molecular mechanisms which regulate the cellular levels of this enzyme. A procedure for isolation of a high yield of total undegraded cellular polysomes was developed. The crosslinking reagent, dimethyl suberimidate, was employed to prepare a stable NADP-GDH-crosslinked-Sepharose-4B antigen affinity column for the purification of rabbit anti-NADP-GDH IgG. Binding studies with ¹²⁵I-labelled antibody and total polysomes, isolated from induced and uninduced cells, showed that the NADP-GDH was being synthesized on polysomes from both types of cells. When poly(A)- containing RNA was extracted from polysomes isolated from induced and uninduced cells, and translated in an mRNA-dependent in vitro translation system, NADP-GDH antigen was synthesized from the RNA from both sources. Based on sucrose density gradient analysis, Chlorella NADP-GDH mRNA has a sedimentation coefficient of 18 Comparison of the amounts of NADP-GDH synthesized in vitro from poly(A)-containing RNA and non-poly(A)-containing RNA showed the NADP-GDH mRNA contained polyadenylic acid sequence. By use of an indirect immunoadsorption procedure, the NADP-GDH mRNA was purified five- to sevenfold from total poly(A)-containing RNA. The overall purification of the NADP-GDH mRNA from total polysomal RNA was approximately two hundred-fold. Complementary DNA was synthesized from the partially purified RNA with reverse transcriptase. The cDNA sequences hybridized to the least abundant class of mRNA sequences present in total poly(A)-containing RNA. In vitro translation of total poly(A)-containing RNA showed that NADP-GDH synthesis was 0.1% of total protein synthesis. Upon addition of inducer to previously uninduced, synchronous cells, the amount of translatable NADP-GDH mRNA increased in a linear fashion after 30 min of the induction period. A change in rate of NADP-GDH mRNA accumulation was observed after 30 min of the induction period. The results support the prediction that since the NADP-GDH enzyme is unstable in vivo, during periods of NADP-GDH accumulation, the NADP-GDH mRNA accumulates. When poly(A)-containing RNA, isolated from uninduced synchronous cells was translated in vitro, NADP-GDH antigen was synthesized at each time in the cell cycle examined. The amount of translatable NADP-GDH mRNA increased throughout the cell cycle with a rate change occuring during the S-phase. This pattern of NADP-GDH mRNA accumulation is consistent with the hypothesis that NADP-GDH mRNA accumulates in uninduced cells at a rate proportional to gene dosage. These results provide one explanation for the observed pattern of enzyme potential in synchronous cells cultured in the absence of inducer. The data are consistent with the possibility that a single mRNA, which is subject to post-transcriptional modification by the inducer, codes for NADP-GDH. / Ph. D.

A new five-step purification procedure was developed which could be completed in five days with an 80 to 85 percent yield of electrophoretically pure nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana. A monospecific, highly purified antibody against the enzyme was prepared from the antisera of immunized rabbits. The antibody was purified on a stable antigen affinity column which was prepared by covalent-coupling of the holoenzyme to CNBr activated Sepharose 4B followed by cross-linking of the enzyme subunits together with dimethylsuberimidate. With ³H-leucine and ³⁵S-sulfate, as labeling agents of whole cells, the reproducibility and efficiency of both direct- and indirect-immunoprecipitation procedures were tested with pure labeled-enzyme as an internal standard. For these studies, a partially purified sheep anti-rabbit immunoglobulin G fraction was employed as a secondary antibody. A modified indirect immunoprecipitation procedure was tested in a cell cycle experiment to determine whether it would be feasible to use as a tool for further cell cycle experiments on enzyme turnover. Although the method was somewhat lengthy, it appeared to be reliable and was used to reveal that during the first one-third of the cell cycle of fully-induced cells, the NADPGDH has a maximum half-life of 1.6 h. Rocket immunoelectrophoresis was used to show that enzyme catalytic activity and antigen increased in a parallel fashion during the cell cycle of fully-induced cells. These data suggest that the cell cycle catalytic activity pattern reflects de novo synthesis of new molecules of enzyme. Since the enzyme in fully-induced cells undergoes rapid in vivo degradation, changes in the rate of enzyme synthesis probably determine the rate of enzyme accumulation during the Chlorella cell cycle in the continuous presence of ammonium. / Ph. D.

Soluble horseradish peroxidase (HRP) is internalized by Chinese hamster ovary cells, a cell line of fibroblastic origin (Adams et al., 1982). We have confirmed this result by showing no inhibition of uptake in the presence of divalent cation chelators (EGTA Mg or EDTA), excess (19 mg/ml) yeast mannan (an inhibitor of uptake through a mannose/N-acetylglucosamine receptor) or using periodate treated HRP. Periodate treatment destroys the ring structure of sugars on HRP which have hydroxyl groups on adjacent ring carbons, eliminating sugar mediated uptake of HRP. Once internalized, HRP is found in endocytic vesicles which by HRP-cytochemical staining, show deposits which rim the luminal face of vesicle membrane. Once HRP is in lysosomes, cytochemical deposits are luminal. To test if HRP is actually associated with vesicle membrane, a hypotonic lysis assay was used. Postnuclear supernatants (PNS) from cells pulse labeled with HRP were lysed and the percent of HRP sedimenting with a high speed membrane fraction was used as a measure of membrane association. After a pulse, >60% of the total HRP internalized was pelletable. Hypotonic lysis of a PNS at different pH and temperature showed no significant difference in "pelletability" from 4℃ to 37℃ at neutral pH and only a slight decrease in "pelletability" with increased temperature (4℃ to 37℃) at pH M.6. Binding of HRP in a membrane preparation was pH and temperature stable. Uptake of native HRP in the presence of yeast mannan (19 mg/ml) or using periodate treated HRP also had little effect on "pelletability", suggesting the absence of sugar specific binding in endocytic vesicles. Using the hypotonic lysis assay of a PNS after different chase times, HRP dissociation from membrane was observed over a 30 minute chase period. Internalized HRP in the presence of yeast mannan (19 mg/ml), intravesicular pH elevators HEPES (40 mM) or monensin (10 μM), or substances which should deplete cellular ATP NaF/KCN (2 mM /1 mM), showed no inhibition of dissociation kinetics. A chase at 17℃ inhibited dissociation of HRP over the entire 30 minute period. This HRP binding site(s) appears unique to endocytic vesicles. A minimum of four steps in transport have been identified based on their sensitivity to inhibitors. HRP transport, identified by Percoll density gradient fractionation, was inhibited at 17°C and was sensitive to pH elevators (NH₄Cl, monensin, HEPES) and ATP depletion (NaF/KCN). Inhibition of transport appeared to be independent of HRP dissociation except at early temperature sensitive step(s). These results suggest that transport inhibition may be due to an effect on a) inhibition of membrane dissociation (early step(s)) and alteration of membrane fluidity (later steps) by reduced temperature and b) transmembrane events by pH elevators and ATP depletion. / Ph. D. / incomplete_metadata

In the round worm C. elegans, it has recently been shown that autophagy, a highly conserved lysosomal degradation pathway that is present in all eukaryotic cells, is required for maintaining healthspan and for increasing the adult lifespan of worms fed under dietary restriction conditions or with reduced IGF signaling. It is currently unknown how extracellular signals regulate autophagy activity within different tissues during these processes and whether autophagy functions cell-autonomously or nonautonomously. We have data that for the first time shows autophagy activity in the neurons and intestinal cells plays a major role in regulating adult lifespan and the longevity conferred by altered IGF signaling and dietary restriction, suggesting autophagy can control these phenotypes cell non-autonomously. We hypothesize that autophagy in the neurons and intestinal cells is an essential cellular process regulated by different signaling pathways to control wild type adult lifespan, IGF mediated longevity and dietary restriction induced longevity. Excitingly we also have found that in animals with reduced IGF signaling autophagy can control longevity in only a small subset of neurons alone. Autophagy in either specific individual chemosensory neurons or a small group of them is completely sufficient to control IGF mediated longevity. This work provides novel insight to the function and regulation of autophagy which will help shed light on understanding this essential process in higher organisms, including mammals. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

The main goal of this thesis work was to develop, implement and evaluate an algorithm that enables mining association rules from datasets that contain quantified distance information among the items. This was accomplished by extending and enhancing the Apriori Algorithm, which is the standard algorithm to mine association rules. The Apriori algorithm is not able to mine association rules that contain distance information among the items that construct the rules. This thesis enhances the main Apriori property by requiring itemsets forming rules to“deviate properly" in addition to satisfying the minimal support threshold. We say that an itemset deviates properly if all combinations of pair-wise distances among the items are highly conserved in the dataset instances where these items occur. This thesis introduces the notion of proper deviation and provides the precise procedure and measures that characterize it. Integrating the notion of distance preserving frequent itemset and proper deviation into the standard Apriori algorithm leads to the construction of our Distance-Based Association Rule Mining (DARM) algorithm. DARM can be applied in data mining and knowledge discovery from genetic, financial, retail, time sequence data, or any domain where the distance information between items is of importance. This thesis chose the area of gene expression and regulation in eukaryotic organisms as the application domain. The data from the domain was used to produce DARM rules. Sets of those rules were used for building predictive models. The accuracy of those models was tested. In addition, predictive accuracies of the models built with and without distance information were compared.

Abstract not available