This thesis presents two separate pieces of work pertaining to pre-mRNA splicing in mammalian cells. The first part examines the regulation of the alternative splicing of the PKM gene in cancer cells, while the second part investigates the physical connections between the transcriptional apparatus and splicing factors. Cancer cells uniformly alter key aspects of their metabolism, including their glucose usage. In contrast to quiescent cells, which use most of their glucose for oxidative phosphorylation when oxygen is present, under the same conditions, most of the glucose consumed by cancer cells is converted to lactate. This phenomenon is known as aerobic glycolysis, and is critical for cancer cell growth. The pyruvate kinase isoform expressed by the cell is a key determinant of glucose usage. Pyruvate kinase in most tissues is produced from the PKM gene, which is alternatively spliced to produce to produce the PKM1 or PKM2 isoforms, which contain exons 9 or 10 respectively. Adult tissues express predominantly the PKM1 isoform, which is universally reverted to the embryonic PKM2 isoform in cancer cells. PKM2 expression promotes aerobic glycolysis. In Chapter 3, I describe a mechanism by which cancer cells promote switching to PKM2. We show that PKM exon 9 is flanked by binding sites for the RNA-binding proteins hnRNP A1/A2 and PTB. These proteins bind to exon 9 and repress its inclusion in the mRNA, resulting in PKM2 production. Additionally, we show that hnRNP A1/A2 and PTB are all overexpressed in cancers in a way that precisely correlates with the expression of PKM2. Finally, we show that the oncogenic transcription factor c-Myc promotes PKM2 expression by transcriptionally upregulating the genes encoding hnRNP A1/A2 and PTB. In the second part of my work, presented in Chapter 5, I examine the coupling of transcription and splicing. The RNA polymerase II C-terminal domain (CTD) plays an important role in ensuring that pre-mRNA transcripts are efficiently spliced, most likely through interactions between splicing factors and the CTD. We have established a biochemical complementation system that has facilitated the identification of a splicing factor that binds to the CTD. Surprisingly, purification of the factor revealed it to be a complex containing U2AF65 and the Prp19 complex, two central splicing factors that had not previously been shown to interact. This complex is functional: I present evidence that the two factors can only activate splicing of the IgMA3 pre-mRNA when they are engaged in a complex. I go on to show that U2AF65 binds directly to the CTD, and this interaction stimulates the RNA binding of U2AF65.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder characterized by a progressive loss of cognition. Histopathologically, AD is defined by the presence of two lesions, senile plaques (SP) and neurofibrillary tangles (NFT), which result from the accumulation and deposition of the amyloid-β peptide (Aβ) and the aggregation of hyperphosphorylated tau protein, respectively. Aβ is formed upon sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases and is secreted extracellularly. The accumulation of extracellular Aβ is thought to initiate a pathogenic cascade resulting in synaptic dysfunction in neurons, followed by the their eventual demise through apoptosis. However, while Aβ has been shown to be increased in AD patients' brains, little is known about how the cleavage of APP and the subsequent generation of Aβ is influenced or if the cleavage process changes over time. Moreover, while the effects of Aβ on neurons are known, the exact mechanism remains unclear. Many have postulated that Aβ exerts its effects by binding a putative receptor, but the search for an Aβ receptor has so far remained inconclusive. Interestingly, one of the proposed potential receptor for Aβ is APP itself. In this model, soluble oligomeric Aβ binds cell-surface APP, inducing its dimerization leading to all the downstream effects of Aβ in cells -- e.g. cell death and/or synaptic dysfunction. Moreover, it has been proposed that Aβ can promote its own production in neurons, thereby initiating a pathogenic loop. However, isolating Aβ-induced APP signaling has remained challenging due to the promiscuous nature of Aβ binding. To work around this problem, we used an antibody-mediated approach to artificially trigger the dimerization of cell-surface APP in cells. We found that dimerization of APP could recapitulate all of the effects of oligomeric Aβ in hippocampal neurons, triggering neuronal death at high concentrations and interfering with normal synaptic functions low concentrations. We also found that dimerization of APP is sufficient to promote the amyloidogenic pathway, by increasing levels of the β-secretase BACE1, resulting in increased Aβ production. Finally, we found that dimerization of APP triggered caspase-dependent cleavage of APP and the formation of a second neurotoxic fragment, termed C31, which also mimics the effects of Aβ in hippocampal neurons. Taken together, our data provides support for the occurrence of a positive pathogenic feedback loop involving Aβ, APP and C31 in neurons.

In the following chapters, I describe factors underlying actin cable dynamics and assembly in the budding yeast, S. cerevisiae. First, I examined the role of type II myosin and a tropomyosin isoform in retrograde actin flow (Chapter II). In yeast and other cell types, actin undergoes retrograde or centripetal movement from the cell cortex towards the interior of the cell. Retrograde actin flow drives intracellular and cellular movement. Previous work in the Pon laboratory showed that actin cables undergo retrograde flow, which occurs, in part, from the force generated from actin polymerization and assembly at the elongating filament end. First, we find that the type II myosin, Myo1p, facilitates retrograde flow. We found that the rate of retrograde actin cable flow is reduced by 1) deletion of Myo1p, 2) displacement of Myo1p from the bud neck, or 3) a conditional mutation that inhibits Myo1p motor activity. These findings indicate that myosin motor activity provides the pulling force to drive movement of elongating actin cables from their site of assembly in the bud tip toward the mother cell. Additional work found that a tropomyosin isoform, Tpm2p, negatively regulates retrograde flow through inhibition of type II myosin binding to F-actin within actin cables. Since type II myosins and tropomyosins have a similar function in retrograde actin flow in animals cells, these findings provide the first evidence that yeast can be used as a model system to study this fundamental, conserved mechanism for actin dynamics. Second, I conducted a drug-based screen for novel regulators of actin cables (Chapter III). Previous studies revealed a role for the yeast formins (Bni1p and Bnr1p) in stimulating polymerization of F-actin for actin cable formation, elongation and retrograde flow, and for other actin cable constituents including tropomyosins and actin bundling proteins in stabilizing and organizing F-actin within actin cables. Earlier work has revealed both that actin cables are selectively destabilized by low levels of the actin-destabilizing drug Latrunculin-A (Lat-A), and this drug inhibits cell growth. I carried out a screen designed to identify non-essential gene deletions that reduce the sensitivity of yeast to the growth inhibiting effects of low doses of Lat-A. Eighteen out of 4,848 deletion strains comprising the yeast deletion library exhibited reduced sensitivity to low levels of Lat-A. Eight of the genes represent uncharacterized open reading frames (ORFs) or encode proteins with no known function or activity. Deletion of a majority of these gene results in increased actin cable number. Additionally, I found the growth inhibiting effects of Lat-A are not suppressed by 1) overexpression of either of TPM1 or TPM2 or 2) deletion of TPM2 and the associated increase in the rate of retrograde actin cable flow. Moreover, I found that one of the genes that reduces the growth-inhibiting effects of Lat-A, YHR022c, is an uncharacterized ORF which encodes a novel Ras-like protein. We call this gene Rar1p for Ras-like actin cable regulator. I found that deletion of RAR1 or expression of a constitutively active formin (Bni1p) produces similar phenotypes: 1) increased actin cable content in the presence and absence of low levels of Lat-A, 2) increased retrograde actin cable flow rates, and 3) resistance to Lat-A-dependent inhibition of growth. Finally, I found that the increase in actin cable content observed upon deletion of RAR1 requires Bni1p and not Bnr1p. Our findings reveal a role for previously uncharacterized genes in the regulation of actin cable stability, and new roles for previously characterized, conserved genes in this process. Equally important, I identified a novel Ras-like protein, Rar1p, and found that it affects actin cable abundance and sensitivity to Lat-A by functioning as an isoform-specific, negative regulator of the formin protein Bni1p. Chapter IV describes future directions for the work outlined in chapters II and III.

This thesis presents two separate pieces of work pertaining to pre-mRNA splicing in mammalian cells. The first piece, as the main research project of the thesis, consists of two related parts. The first part identified the regulators of the alternative splicing of the PKM gene in cancer cells while the second part elucidates the molecular mechanism of how this mutually exclusive alternative splicing is regulated. The second piece investigates the molecular mechanism of how SRp38 functions as a splicing activator when phosphorylated. Cancer cells uniformly alter key aspects of their metabolism, including their glucose usage. In contrast to quiescent cells, which use most of their glucose for oxidative phosphorylation when oxygen is present, under the same conditions, most of the glucose consumed by cancer cells is converted to lactate. This phenomenon is known as aerobic glycolysis, and is critical for cancer cell growth. The pyruvate kinase isoform expressed by the cell is a key determinant of glucose usage. Pyruvate kinase in most tissues is produced from the PKM gene, which is alternatively spliced to produce the PKM1 or PKM2 isoforms, which contain exons 9 or 10 respectively. Adult tissues, such as skeletal muscle and brain, express predominantly the PKM1 isoform, which is universally reverted to the embryonic PKM2 isoform in cancer cells. PKM2 expression promotes aerobic glycolysis. In Chapter 3, I describe a mechanism by which cancer cells promote switching to PKM2. We show that PKM exon 9 is flanked by binding sites for the RNA-binding proteins hnRNP A1/A2 and PTB. These proteins bind to exon 9 and repress its inclusion in the mRNA, resulting in PKM2 production. Additionally, we show that hnRNP A1/A2 and PTB are all overexpressed in cancers in a way that precisely correlates with the expression of PKM2. Finally, we show that the oncogenic transcription factor c-Myc promotes PKM2 expression by transcriptionally upregulating the genes encoding hnRNP A1/A2 and PTB. In Chapter 4, I provide additional insights into how PKM AS is regulated and a novel discovery that general splicing repressors can repress either one of the two mutually exclusive exons at different expression levels, through protein-protein interactions of these proteins bound on different sets of binding sites on and flanking each. First, using a splicing minigene construct that recapitulates PKM splicing in HeLa cells, we identified additional PTB and hnRNP A1/ A2 ISSs in intron 9 necessary for full exclusion of exon 9. More importantly, we found two ESSs in exon 9, absent from exon 10, that match the hnRNP A1 consensus, and which are critical for exon 9 exclusion. We show that these ESSs function cooperatively to facilitate hnRNP A1 binding to an intronic splicing silencer in intron 9 described in Chapter 3. I also elucidated the mechanism of how exon 10 is excluded when exon 9 is derepressed and show that hnRNP A1 and PTB, when their protein levels are reduced, release the inhibition of exon 9 but repress exon 10 inclusion, through binding sites present in introns 9 and 10. This mechanism, coupled with nonsense mediated decay, function to prevent the appearance of PKM mRNA containing both exon 9 and exon 10. In the second piece of work, presented in Chapter 5, I, based on the findings from a previous post doctor that SRp38 functions as a sequence-specific splicing activator, showed that SRp38 promotes spliceosomal complex A formation. I examined the mechanism of spliceosomal A complex formation and found that SRp38 promotes the recruitment of U1 and U2 snRNPs to splicing substrates that contain high-affinity SRp38 binding sites.

The production of retroviral enzymes requires a translational recoding event which subverts normal decoding, either by direct suppression of termination with the insertion of an amino acid at a stop codon (readthrough), or by an alteration of the reading frame of the mRNA (frameshift). It has been determined that retroviral readthrough and frameshift require cis-acting factors in the mRNA to stimulate recoding on the eukaryotic ribosome. Here we investigate the affects of trans acting factors on recoding, primarily in the context of the MoMLV gag-pol junction. We report the effects of a host protein, Large Ribosomal Protein Four (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we show that transfection of cells with an RPL4 cDNA expression construct enhances recoding efficiency in a dose-dependent manner. The increase in the frequency of recoding can be more than 2-fold, adequate to disrupt normal viral production. This effect is cell line specific, and appears to be distinct to RPL4 among ribosomal proteins. The RPL4 increase occurs with both retroviral readthrough and frameshift sequences, and even at other viral readthrough regions that do not involve RNA secondary structures. We show that RPL4 effects are negated by release factor over-expression, and that RPL4 will increase readthrough above the levels of a hyperactive mutant and in addition to G418. When cotransfected with Moloney murine leukemia provirus, the RPL4-mediated increase in readthrough reduces the amount of virus released. We also examined the effects of aminoglycoside drugs and the small molecule PTC124 on readthrough of the MoMLV gag-pol junction. We show that G418, paromomycin and PTC124 increase readthrough of our MoMLV reporter in a dose dependent manner in 293A cells. These drugs reduce viral replication, as measured by a recombinant transducing virus assay. We further examine G418 and paromomycin in an in-vitro system; readthrough is increased to higher levels than those seen in vivo. G418 displays deleterious effects on cell viability and overall translation. Paromomycin does not appear as toxic, suggesting differences in interactions by which these drugs enhance readthrough.

Movement and positional control of mitochondria and other organelles are coordinated with cell cycle progression in the budding yeast, Saccharomyces cerevisiae. Recent studies have revealed a checkpoint that inhibits cytokinesis when there are severe defects in mitochondrial inheritance. An established checkpoint signaling pathway, the mitotic exit network (MEN), participates in this process. Here, we describe mitochondrial motility during inheritance in budding yeast, emerging evidence for mitochondrial quality control during inheritance, and organelle inheritance checkpoints for mitochondria and other organelles.

In the morphogenesis of tissue development, how coordination of patterning and growth achieve the correct organ size and shape is a principal question in biology. Efficient orchestrating mechanisms are required to achieve this and cells have developed sophisticated systems for reception and interpretation of the multitude of extracellular stimuli to which they are exposed. Plasma membrane receptors play a key role in the transmission of such signals. G-protein coupled receptors (GPCRs) are the largest class of cell surface receptors that respond to an enormous diversity of extracellular stimuli, and are critical mediators of cellular signal transduction in eukaryotic organisms. Signaling through GPCRs has been well characterized in many biological contexts. While they are a major class of signal transducers, there are not many defined instances where GPCRs have been implicated in the process of development to date. The Drosophila wing provides an ideal model system to elucidate and address the role of GPCRs in development, as its growth is regulated by a small number of conserved signaling pathways. In my thesis work, I address the role of a trimeric G alpha protein in Drosophila, Gαf, and what part it may play in development. In particular, I explore the role of Gαf as an alpha subunit of a trimeric complex, to determine what heptahelical receptors might act as its cognate receptor.

Obesity is suspected to be a risk factor for aggressive PC due to its associations with altered circulating levels of metabolic and sex steroid hormones involved in prostate development as well as oncogenesis. However, the current observational evidence linking obesity to aggressive PC is inconsistent or conflicting, and there is growing concern that much of the heterogeneity across studies may be the result of obesity interfering with PC screening, diagnosis, and treatment. We performed a critical review of studies analyzing the association between anthropomorphic measures and overall PC risk, as well as risk of aggressive disease, and illustrate how unique aspects of PC diagnosis and treatment render its risk factor associations unusually susceptible to selection biases which are largely unabated by conventional statistical adjustment. Using a counterfactual framework to describe the selection processes that give rise to these biases, we demonstrate instances in which the use of marginal structural models (MSM) and inverse probability weighting (IPW) may be able to address such biases. Using data collected on a series of patients referred for prostate biopsy, and found to have PC, we examined the association between BMI, clinical and pathological characteristics. We found evidence of differential receipt of radical prostatectomy (RP) by BMI category, and history of obesity which, in the latter case, partially attenuated the association between obesity and high grade biopsy. After multivariate statistical adjustment and IPW, obesity was associated with increased odds of higher pathological grade and stage after RP, associations which were not apparent without the use of IPW. We also examined the association between one's exposure to history of obesity (measured at age 20, 40 and near the time of diagnosis), and found that men with a BMI ≥30 at all three measures had an increased odds of high pathologic stage (≥pT3), tumor volume >30mm3, and positive surgical margins, compared to never obese. In the multivariate models which did not use inverse probability weights, only the association between chronic obesity and high pathological grade reached statistical significance. These findings suggest that treatment selection factors caused a bias toward the null in our estimates of the associations between history of obesity and adverse tumor characteristics, and would have substantively altered the overall findings of the study. We then conducted multiplex immunoflorescence immunohistochemistry on tissue microarrays (TMA) made from representative cores of tumor tissue from RP specimens. Using a semi-automated, florescence microscopy and imaging technique, we measured nuclear expression or androgen receptor (AR), epithelial insulin like growth factor I receptor (IGF-IR), and proliferation marker Ki67, in 357 cases who received a RP. We then tested for associations between patient history of obesity and other demographic and clinical characteristics. Expression of AR and Ki67 were positively associated with tumor grade and stage, while Ki67 and IGF-IR were associated with tumor volume in excess of 30mm3. We also found an inverse association between IGF-IR and tumor grade. We did not, however, find that history of obesity was significantly associated with expression of any of the biomarkers. Thus we have found no evidence that the association between chronic obesity and aggressive disease is mediated by differential expression of androgen or IGF-I receptor, or greater tumor proliferation (Ki67). As researchers continue to understand the underlying causes of aggressive PC and pursue the goal of personalized medicine, studies such as these become increasingly important as they have the potential to reduce the biases inherent in these dataset and explore important interactions between risk factors, and tumor phenotypes that may point the way to new preventive and treatment.

This dissertation serves to analyze the molecular and cellular signaling mechanisms by which aldose reductase (AR) contributes to ischemia/reperfusion (I/R) injury, specifically in the myocardium. It provides a comprehensive overview of the pathophysiology associated with acute coronary syndromes, the mechanisms of normal myocardial substrate metabolism, and how metabolism is altered under ischemic conditions. Specific chapters in this dissertation aim to elucidate whether AR pathway mediated I/R injury is linked to changes in glycogen synthase kinase 3β (GSK3β) phosphorylation, and whether an increased flux via AR and resultant I/R injury is mediated by changes in autophagy.

Sarcomas are cancers of connective tissues, such as bone, adipose and cartilage, and are thought to arise from the aberrant development of the mesenchyme. As such, mesenchymal stem cells are thought to be the cell of origin for sarcomas. Genetic or epigenetic lesions at particular points during the differentiation of a mesenchymal stem cell into its terminal mesenchymal cell type are able to give rise to specific subtypes of sarcomas. Recently, a number of reports have identified elevated expression of the human Piwi homolog--called Hiwi--in a variety of human cancers, including gastric cancer, pancreatic cancer, gliomas and, most relevant for this dissertation, sarcomas. In sarcomas, Hiwi is highly expressed and elevated Hiwi prognosticates shorter patient survival. Hiwi is the human homolog of the Piwi family of proteins, which are members of the Paz-Piwi Doman (PPD) family. During normal development, Piwis are thought to maintain stem cells of the germline, and indeed their expression is limited to early development and to the adult germline. Piwis are thought to maintain stem cells in the germline with small RNA partners, called piwi-interacting RNAs (piRNAs). More specifically, Piwi/piRNA complexes in the germline are thought to maintain transposon silencing, and thus ensure genomic stability. A detailed mechanism by with Piwis suppress transposon migration in the germline remains an area of active investigation, but is thought to occur via DNA methylation of transposon regions. In this way, Piwis are critical for maintenance of genomic integrity of germline stem cells during normal development. Thus, the finding that Piwis are elevated in human cancers is directly in conflict with its known role in ensuring genomic stability during development. Piwi homologs are critical for maintenance of germline stem cells during development but aberrant Hiwi expression has also been identified in all cancers examined, including in sarcomas. A potential connection between mesenchymal stem cells, sarcomas and Hiwi remains unexplored. Moreover, the role of Hiwi in sarcomas is unknown. In the studies presented here, we demonstrate that over-expressing Hiwi in mesenchymal stem cells inhibits their differentiation in vitro and generates sarcomas in vivo. Secondly, transgenic mice expressing Hiwi (mesodermally-restricted) develop sarcomas. Conversely, inducible down-regulation of Hiwi in human sarcomas inhibits growth and re-establishes differentiation. These data reveal that Hiwi is directly tumorigenic. We have also identified the presence of piRNAs in our Hiwi-expressing models. We further show that DNA methylation correlates with Hiwi expression and that cyclin-dependent kinase inhibitor (CDKI) tumor suppressor genes are silenced upon Hiwi over-expression. Moreover, Hiwi's tumorigenic effects are reversible using DNA de-methylating agents. These studies reveal for the first time not only a novel oncogenic role for Hiwi as a driver of tumorigenesis, but also suggest that the use of epigenetic agents may be clinically beneficial for treatment of tumors that express Hiwi. Additionally, our data showing that Hiwi-associated DNA hyper-methylation with subsequent genetic and epigenetic changes favoring a tumorigenic state reconciles the conundrum of how Hiwi may act appropriately to promote genomic integrity during early development (via transposon silencing) and inappropriately in adult tissues with subsequent tumorigenesis.