Splicing of mRNA precursors (pre-mRNA) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use in splicing is not well understood. Several yeast splicing factors belong to DEAD/H box family of RNA-dependent ATPase, and are implicated in dynamic RNA structure rearrangement during spliceosome assembly. In mammals, however, such information is conspicuously lacking. In fact, none of the known mammalian splicing factors has characteristics for ATP hydrolysis. In an attempt to identify mammalian splicing factors involved in ATP usage, we have developed a novel approach to identify and purify spliceosomal ATP binding proteins. Six spliceosomal ATP binding proteins were found, one of them, SAFp56, was purified and microsequenced, and found to be a DEAD box protein containing unique DECD motif instead of the canonical DEAD motif. During the course of this work, a new functional region in U2AF65, an essential splicing factor required for U2 snRNP entry into the spliceosome, was defined. This information was used to clone a human U2AF65 associated protein (UAP). UAP and SAFp56 are identical. We refer to this protein as hUAP56 (human 56 kDa U2AF65 associated protein). We present evidence that hUAP56 is an essential splicing factor required for the U2 snRNP binding to pre-mRNA. Interestingly, UAP56 is recruited to pre-mRNA in a polypyrimidine tract bound U2AF65-dependent fashion. This result underscores a new function of U2AF65, and provides the first description of how a specific DEAD box protein is directed to a pre-mRNA splicing signal, and/or, to the proximity of its substrate at a particular stage. Like an authentic DEAD box protein. hUAP56 has ATP binding, RNA-stimulated ATPase, as well as RNA binding activity. A particularly novel result is that the ATPase activity of UAP56 is stimulated by U2AF65. This observation strongly suggests the role of UAP56 in ATP dependent mechanism during U2 snRNP binding to the pre-mRNA branchpoint, and implies that UAP56 may function through a distinct mechanism. We identify yeast UAP (yUAP), a highly conserved S. cerevisiae homologue of hUAP56. yUAP is essential for viability, can be functionally substituted for by hUAP56, and like its human counterpart, is an essential pre-mRNA splicing factor required for spliceosome assembly. Furthermore, we show that yUAP is required for formation of the branchpoint dependent commitment complex, the precursor for U2 snRNP addition. Site-directed mutagenesis revealed that all DEAD box protein consensus motifs are required for yUAP function. Interestingly, a strain harboring a yUAP mutant in which the DECD sequence, characteristic of UAP members, was changed to canonical sequence, is inviable. Our results demonstrate that UAP is structurally and functionally conserved from yeast to man. In conjunction with previous studies, we conclude that at least two DEAD box proteins, Prp5p and yUAP, are required for the U2 snRNP-branchpoint interaction.

Disruption of cilia proteins results in a range of disorders called ciliopathies. However, the mechanism by which cilia dysfunction contributes to disease is not well understood. Intraflagellar transport (IFT) proteins are required for ciliogenesis. They carry ciliary cargo along the microtubule axoneme while riding microtubule motors. Interestingly, IFT proteins localize to spindle poles in non-ciliated, mitotic cells, suggesting a mitotic function for IFT proteins. Based on their role in cilia, we hypothesized that IFT proteins regulate microtubule-based transport during mitotic spindle assembly. Biochemical investigation revealed that in mitotic cells IFT88, IFT57, IFT52, and IFT20 interact with dynein1, a microtubule motor required for spindle pole maturation. Furthermore, IFT88 co-localizes with dynein1 and its mitotic cargo during spindle assembly, suggesting a role for IFT88 in regulating dynein-mediated transport to spindle poles. Based on these results we analyzed spindle poles after IFT protein depletion and found that IFT88 depletion disrupted EB1, γ-tubulin, and astral microtubule arrays at spindle poles. Unlike IFT88, depletion of IFT57, IFT52, or IFT20 did not disrupt spindle poles. Strikingly, the simultaneous depletion of IFT88 and IFT20 rescued the spindle pole disruption caused by IFT88 depletion alone, suggesting a model in which IFT88 negatively regulates IFT20, and IFT20 negatively regulates microtubulebased transport during mitosis. Our work demonstrates for the first time that IFT proteins function with dynein1 in mitosis, and it also raises the important possibility that mitotic defects caused by IFT protein disruption could contribute to the phenotypes associated with ciliopathies.

Endometrial prostaglandins (PGs) play a role in menstruation in primates and in luteolysis in nonprimates. Their biosynthesis is regulated by estrogen (E) and progesterone (P) in a manner not fully understood. The purpose of this thesis research was to (1) study the effects of E and P, both in vivo and in vitro, on basal endometrial PG output in vitro during the course of the artificial menstrual cycle in the rhesus monkey, and (2) further to examine the cellular mechanisms of P action in vivo on PG output using an ovine model system. To carry out the first objective, ovariectomized rhesus monkeys (n=39) were maintained on either a standard or manipulated artificial menstrual cycle (SAMC and MAMC, respectively) and endometrial biopsies were obtained at precise times in separate cycles on: cycle day 9 (mid-proliferative), 13 (mid-cycle E peak), 14 (one day post E peak), and 23 (mid-secretory). PGF2α was the most abundant PG produced in vitro by endometrial organ cultures, the levels of which changed most dramatically throughout the SAMC. Within the first 24 hours of organ culture, PGF2α accumulation was low on day 9 and rose significantly (p<0.01) on day 13, indicating a stimulatory effect of E in vivo. However, E added in vitro, at either physiologic or supraphysiologic concentrations, to endometrial cultures did not stimulate PGF2α accumulation on any cycle day examined. On day 14, just one day post E peak, there was a dramatic fall in PGF2α accumulation which appeared to be due to both a decline in stimulatory E in vivo and a rise in inhibitory P in vivo. Basal PGF2α accumulation in vitro by day 23 endometrial cultures was 10-fold higher (p<0.01) compared to days 9 and 14. This high level of PGF2α output on day 23 appeared to be caused by a paradoxical priming effect of P in vivo and also a slight enhancement by the mid-cycle peak of E in vivo. Padded in vitro, at a physiologic concentration, to day 23 endometrial cultures markedly inhibited (p<0.01) the high level of PGF2α accumulation, suggesting that P withdrawal in vivo promotes the rise in endometrial PGF2α production in vivo at the time of menstruation in primates. An ovine model system was further used to investigate the cellular mechanisms of P action in vivo. Ovariectomized sheep (n=8) were administered an infusion regimen of either E and P, or E and P vehicle alone, to examine the effects of P in vivo on PGF2α production in vitro by endometrial explants during short-term incubations. P in vivo increased the mass amount of stimulated PGF2α output by both physiologic and pharmacologic mechanisms. In addition, P did not appear to significantly alter the sensitivity of the endometrium to stimulatory levels of oxytocin in vitro indicating that the cellular events accounting for the P priming effect, in part, may occur independent of the oxytocin receptor closer to the PG biosynthetic pathway. In P-primed endometrium, the mass amount of PGF2α stimulated by a calcium-ionophore (A23l87) was less than that stimulated by OT suggesting the involvement of calcium-insensitive mechanisms in PGF2α synthesis.

Circadian rhythms are self-sustained 24-hour period oscillations present in most organisms, from bacteria to human. They can be synchronized to external cues, thus allowing organisms to anticipate environmental variations and optimize their performance in nature. In Drosophila, the molecular pacemaker consists of two interlocked transcriptional feedback loops. CLOCK/CYCLE (CLK/CYC) sits in the center and drives rhythmic transcription of period (per), timeless (tim), vrille (vri) and PAR domain protein 1 (Pdp1). PER and TIM negatively feedback on CLK/CYC transcriptional activity, forming one loop, while VRI and PDP1 form the other by regulating Clk transcription negatively and positively, respectively. Posttranscriptional and posttranslational regulations also contribute to circadian rhythms. Although much has been learned about these feedback loops, we are still far from understanding how stable 24-hour period rhythms are generated. My thesis work was to determine by which molecular mechanisms kayak-α (kay-α) and Ataxin-2 (Atx2) regulate Drosophila circadian behavior. Both genes are required for the precision of circadian rhythms since knocking down either gene in circadian pacemaker neurons results in long period phenotype. The work on kay-α constitutes the first half of my thesis. We found that the transcription factor KAY-α can bind to VRI and inhibit VRI’s repression on the Clk promoter. Interestingly, KAY-α can also repress CLK’s transcriptional activity on its target genes (e.g., per and tim). Therefore, KAY-α is proposed to bring precision and stability to the molecular pacemaker by regulating both transcriptional loops. The second half of my thesis focuses on ATX2, an RNA binding protein whose mammalian homolog has been implicated in neurodegenerative diseases. We found that ATX2 is required for PER accumulation in circadian pacemaker neurons. It forms a complex with TWENTY-FOUR (TYF)—a crucial activator of PER translation—and promotes TYF’s interaction with Poly(A)-binding protein. This work reveals the role of ATX2 in the control of circadian rhythms as an activator of PER translation, in contrast to its well-established role as a repressor of translation. It also further demonstrates the importance of translational regulation on circadian rhythms. Finally, it may help understanding how ATX2 causes neuronal degeneration in human diseases.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.

Clinically relevant concentrations of ethanol modulate the function of a number of ion channel proteins. A fundamental question regarding the effects of alcohol is whether the drug modifies ion channels by directly binding to the protein, indirectly by perturbing the surrounding membrane lipid, or some combination of both. This thesis further characterized ethanol's site of action by examining the effects of ethanol on N-methyl-D-aspartate (NMDA) receptor/channels and large conductance Ca2+-activated K+ (BK) channels at a number of levels using direct electrophysiological methods. In Chapter One, the magnitude of ethanol's inhibition of a number of cloned heteromeric NMDA receptor/channels in the absence or presence of a number of modulators was compared. The rank order of ethanol sensitivity for the subunit combinations studied was NR1b/NR2A > NR1b/NR2B > NR1b/NR2C > NR1b/NR2D. Modulation of the receptor with Mg2+, Zn2+, the glycine antagonist 7-Chlorokynurenic Acid, or after reduction or oxidation of the redox regulatory site did not alter the ethanol sensitivity of heteromeric NMDA receptors. Therefore, the ethanol sensitivity of NMDA receptor/channels is dependent upon which NR2 subunit is present, and ethanol's site of action is unrelated to these modulatory sites on the receptor/channel protein. In Chapter Two, ethanol's site of action at cloned BK channels was characterized using of a number of 1-alkanols. Ethanol, butanol, hexanol, and heptanol reversibly and dose-dependently increased the current carried through BK channels. Longer chain 1-alkanols, such as octanol had no effect on channels. In Chapter Three, the action of ethanol on BK channels reconstituted in a number of model planar bilayers was studied. Ethanol increased the activity of BK channels incorporated in bilayers composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) or PE alone by decreasing the average amount of time channels dwelled in the closed state. There was no significant effect of alcohol on either channel conductance or unitary current. Taken together, these data suggest that ethanol action on BK channels does not require the complex membrane architecture found in native membranes, and does not require freely diffusible cytoplasmic factors or proteins.

The relative contribution of T cell receptor-dependent stimulation versus TcR-independent bystander stimulation in the massive increase in the number of activated proliferating CD8+ T cells seen during acute many acute viral infections is unclear. To determine if this increase was the result of TcR-independent bystander activation and proliferation, anti-viral cytotoxic T lymphocytes were induced in vivo via DNA immunization so that the anti-viral immune response could be examined in the absence of the high levels of cytokines generated during acute infection. After a single immunization with a plasmid encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV) a nearly 2 log10 reduction in viral titers in the spleen was observed 3 days after LCMV infection. After 2 or 3 immunizations a greater that 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. After intracerebral challenge vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. By limiting dilution analysis LCMV-specific CTL precursors were detected in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at days 4 after LCMV challenge. However, the numbers of CTL precursors elicited by DNA vaccination was too low to determine if cytokine-mediated TcR-independent bystander activation and proliferation had taken place. HY-specific TcR-transgenic mice, which have a restricted TcR repertoire, and LCMV-carrier mice, which are tolerant to LCMV, were used to determine the extent of TcR-independent bystander activation and proliferation during acute LCMV infection. LCMV infection of C57BL/6 mice induced CTL that lysed uninfected H-2k and H-2d allogeneic targets, but, LCMV-induced CTL from HY- transgenic mice lysed only the H-2k-expressing cells. The HY-mice generated both anti-H-2k and anti-H-2d CTL in mixed lymphocyte cultures, strongly suggesting that the generation of allospecific CTL during acute LCMV-infection is antigen specific. During the LCMV infection there was blastogenesis of the CDB+ T cell population, but the HY-specific T cells remained small in size, and did not alter their expression of the activation molecules CD44 and MEL-14. In order to examine the potential for bystander stimulation under conditions of a very strong CTL response, T cell chimeras were made between normal and HY-transgenic mice. Even in the context of a normal vicus-induced CTL response, no stimulation of HY -specific T cells was observed, and HY-specific cells were diluted in number by day 9 post-infection. In LCMV-carrier mice in which donor and host T cells could be distinguished by Thy 1 allotypic markers, adoptive transfer of LCMV-immune T cells into LCMV-carrier mice, whose T cells were tolerant to LCMV, resulted in activation and proliferation of donor CDB cells but little or no activation of host CDB+ T cells. These results show that TcR-independent bystander activation of non virus-specific T cells is not a significant component of an anti-viral T cell response and support the hypothesis that the massive polyclonal CTL response to LCMV infection is virus-specific. T cells activated during potent anti-viral immune responses are sensitized to undergo apoptosis after strong TcR-stimulation in a process known as activation-induced cell death (AICD). To determine if T cells, not participating in the immune response were also subject to AICD, LCMV-carrier mice were used. Using TUNEL flow cytometry, it was shown that after reconstitution of Thy 1.2+ LCMV-carrier mice with spleen cells from Thy 1.1+ LCMV-immune mice, the Thy 1.2+ host T cells which were not specific for the virus and did not proliferate in a bystander fashion, were rendered sensitive to TcR-induced apoptosis in vitro. This bystander sensitization to AICD was shown not to be dependent on the continued presence of activated proliferating donor cells during the in vitro culture period. Bystander sensitization to AICD was not the result of an antigen presenting cell defect, but rather was the result of an in vivo conditioning of the T cells themselves. The mechanism of this sensitization was, at least, partially dependent on the ability of host T cells to respond to IFNγ, and on the expression of Fas ligand on the activated, proliferating donor cells. This bystander sensitization to AICD may explain why memory T cell responses are so poor during acute viral infection and can serve as a potential mechanism for virus-induced immunosuppression.

The purpose of this work was to determine whether arachidonic acid and other fatty acids might directly regulate the behavior of ion channels. Arachidonic acid is known to be liberated from plasma membrane phospholipid upon activation of cell surface receptors, and to subsequently act as a precursor to biologically active metabolites. This study was based on the rationale that the liberated arachidonic acid itself was a potential regulator of plasma membrane ion channels. The effects of arachidonic acid and other fatty acids on the behavior of ion channels were examined in two preparations of isolated smooth muscle cells. In both cell types, K+-selective ion channels were activated both by arachidonic acid and by fatty acids that are not converted to metabolites through the cyclooxygenase and lipoxygenase metabolic pathways for arachidonic acid. These results indicate that metabolites of these pathways did not mediate the fatty acid response. Further, fatty acids were effective in cell-free patches of membrane in the absence of nucleotides and Ca++, showing that signal transduction mechanisms requiring these and other cytosolic factors were not required. Such signaling mechanisms include those involving phosphorylation, cyclic nucleotides, GTP-dependent proteins, and the NADPH-dependent cytochrome P450 metabolic pathway. Thus fatty acids themselves appear to directly activate K+ channels, much as they directly activate several enzymes, and may constitute a new class of messenger molecules acting on ion channels. The two preparations of cells used were gastric smooth muscle cells from the toad, Bufo Marinus, and pulmonary artery smooth muscle cells from the New Zealand White Rabbit. In gastric smooth muscle cells, a previously undescribed K+ channel was activated by a variety of fatty acids. This channel exhibited a conductance of approximately 50 pS, weak voltage-dependence, and K+ selectivity. The fatty acid structural features required for activation of this channel were examined by testing numerous fatty acids. Further, the same K+ channel was found to be endogenously active in the presence of Ca++ at the extracellular surface of the membrane. In pulmonary artery smooth muscle cells, fatty acids activated K+ channels of a recognizable large-conductance type that is activated by Ca++ at the intracellular membrane surface. This channel type has been widely studied but has not been reported in this preparation. Characteristic of the large-conductance, calcium-activated K+ (CAK) channel type, the channels activated by fatty acids exhibited a conductance of approximately 260 pS, strong voltage-dependence, K+ selectivity, and activation by low concentrations of Ca++ (10-7-10-6 M) at the cytosolic surface of the membrane. Lastly, these CAK channels were found to be activated by membrane stretch.

Induction of germline (GL) ε transcripts, an essential step preceding immunoglobulin (Ig) isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g. CD40 ligand (CD40L). AP-1 (Fos and Jun), induced transiently by CD40L, binds a DNA element in the mouse GL ε promoter. AP-1 synergizes with Stat6 to activate both the intact GL ε promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL ε promoter. By contrast, C/EBPβ, which transactivates the human GL ε promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not transactivate the human GL ε promoter. Thus, due to selective binding of either AP-1 or C/EBP proteins, induction of GL ε transcripts in mouse and human may be regulated differently. In addition to AP-1, NF-кB activity is also induced by CD40L stimulation in normal B cells. Using GST pulldown assays and coimmunoprecipitation techniques I show that NF-кB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. When Stat6 and NF-кB proteins are co-expressed in human embryonic kidney 293 (HEK 293) cells, an IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4. Furthermore, the same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-кB proteins in IL-4-stimulated B lymphoma cells I.29μ. Consistently, by using nuclear extracts from transfected HEK 293 cells and from I.29μ B cells in electrophoretic mobility shift assays (EMSAs), I show that Stat6 and NF-кB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-кB. I conclude that the direct interaction between Stat6 and NF-кB may provide the basis for synergistic activation of the GL ε promoter. Finally, although mouse GL ε transcripts have a half-life of approximately 100 min, the RNA level continues to increase for up to 24 h and the promoter appears to be active for at least 2 days after B cell activation. These data suggest that induction of AP-1 and NF-кB activities by CD40L, although transient, is required for activation of the mouse GL ε promoter by IL-4-induced Stat6.

The parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection. The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs.