Rapid modulation of gene expression is critical for cells to respond to environmental challenges and initiate developmental programs. Cells employ a variety of mechanisms to achieve tight regulation of gene expression, including post-transcriptional control of active messenger RNA (mRNA) levels by inhibition of translation or by mRNA degradation. While mRNA production via transcription has been extensively characterized, our understanding of how mRNAs are partitioned between an actively translating state and an inactive state is limited. In this thesis I examine the role of a highly evolutionarily conserved protein, the DEAD-box ATPase Dhh1, in mRNA inactivation and turnover in S. cerevisiae.

Previous work from our lab puts Dhh1 at the crossroads of mRNA fate. For example, artificially tethering Dhh1 to an mRNA is sufficient to trigger its degradation. In contrast, in cells compromised in the 5'-3' decay pathway, tethered Dhh1 can no longer direct degradation of the message, yet still possesses the ability to repress its translation. Moreover, ATPase activity of Dhh1 is critical for mRNA localization in the cell, as ATPase-deficient mutants of Dhh1 induce the constitutive formation of mRNA-protein (mRNP) foci known as Processing Bodies (PBs) - enigmatic cellular structures that can direct storage or degradation of mRNAs. However, mechanistically how Dhh1 functions in translation repression and mRNA decay, as well as its role in PB assembly has remained elusive.

In the following dissertation, I further characterize Dhh1 activities in translation repression, mRNA degradation, and PB formation. Using the previously established tethering assay, I identified protein factors that are distinctly required for translational repression or mRNA decay by Dhh1. Furthermore, I discovered that a mutant of Dhh1 that cannot bind to ATP is unable to interact with the Ccr4-NOT deadenylase complex - a major intracellular machine involved in transcriptional regulation and mRNA turnover. Finally, I show that Not1, the major scaffold of the Ccr4-NOT complex, controls Dhh1 localization to PB foci. In summary, my work suggests that the ATPase activity of Dhh1 is regulated in vivo, and this regulation may ultimately determine the fate of an mRNA - whether it is actively translated in the cytoplasm, or delivered to Processing Bodies for degradation or storage.

By repressing translation and promoting mRNA decay, cells are able to modulate gene expression and respond swiftly to changing environmental signals and developmental cues. Although translation, storage and degradation of mRNAs are key steps in the post-transcriptional control of gene expression, how mRNAs transit between these processes remains poorly understood.

During my thesis I functionally characterized the DExD/H box ATPase Dhh1, a critical regulator of the cytoplasmic fate of mRNAs. Using mRNA tethering experiments in yeast, I showed that Dhh1 is sufficient to move an mRNA from an active state to translational repression. In actively dividing cells, translational repression is followed by mRNA decay, however, deleting components of the 5' to 3' decay pathway uncoupled these processes. Interestingly, Dhh1's ability to inactivate an mRNA coincided with its ability to move mRNAs into cytoplasmic processing bodies (P bodies).

I also examined the role of ATP hydrolysis in Dhh1's ability to repress translation and activate mRNA decay. While Dhh1's ATPase activity is not essential for translational inhibition and mRNA decay in dhh1Δ cells, I found that ATP hydrolysis regulates P body dynamics and the release of Dhh1 from these RNA-protein granules. Surprisingly, I found that the presence of a wild-type copy of Dhh1 rescues the abnormal P-body localization of a Dhh1 ATPase-mutant. Additionally, the Dhh1 ATPase mutant no longer reduces mRNA and protein levels when tethered to an mRNA in the presence of a wild-type copy of Dhh1. My results place Dhh1 at the interface of translation and decay controlling whether an mRNA is translated, stored or decayed.

Eukaryotic cells can be distinguished from prokaryotic cells because they posses membrane bound organelles. The presence of organelles in cells allowed cellular processes to be isolated into compartments, thus allowing additional levels of regulation to be applied to simple cellular processes. One of these membrane bound organelles, the nucleus, functions to isolate the cell's DNA from the cytoplasm. Large aqueous pores span the nuclear envelope and determines which molecules can enter and exit the nucleus. These channels are called Nuclear Pore Complexes (NPCs). The NPC is made up of a central core that spans the NE, a nuclear basket structure, fibers that extend into the cytoplasm, and is composed of only ~33 different proteins called Nucleoporins. The NPC exhibits an eight-fold rotation symmetry around the plane of the NE, and nucleoporins are present in eight, or multiples of eight, copies. Although the structure and composition of the NPC is well characterized, the process in which NPCs are inserted into the intact NE of yeast is unknown.

In order to learn more about NPC assembly into the intact NE, I developed an assay to monitor the distribution of old and new nucleoporins in live S. cerevisiae cells. I used the photoconvertable fluorescent protein Dendra to examine a report that new NPC assembly occurs exclusively in daughter buds while old NPCs remain with mother cells. We examined two different Nups and observed new pore formation in both mother and daughter cells, additionally we determined that old pores are inherited by both mother and the daughter cells. We hypothesized that the differences in our observations from the previous report was due to differences experimental technique.

To begin to understand the early events of NPC assembly, we first determined which proteins interact with the essential transmembrane Nup Ndc1. We then constructed conditional mutants of different combinations of these proteins and determined that Nup59 is functionally redundant with the combined element of Pom34 and Pom152. We show that these conditional mutants are not viable and result in mislocalization of core and cytoplasmic Nups. We use the Dendra assay to show that depletion of these elements result in a reversible defect in NPC assembly.

Finally, we examine the role of Nup157 and Nup170 in NPC assembly. A nup157∆ strain conditionally expressing Nup170 results in mislocalization of core and cytoplasmic reporter Nups, but not nuclear Nups. This strain also has a reduced number of NPCs by EM in non-permissive conditions. Cells depleted of Nup170 and Nup157 were examined with the Dendra assay and new protein accumulated in the cytoplasm suggesting a block in NPC assembly. This block is overcome by reintroduction of Nup170. The work presented here represents a new way to study NPC assembly and reveals a few early events in NPC assembly.

Regardless of size, shape, or function, all cells must rapidly respond to a changing environment, especially in adverse conditions. Various environmental stresses including heat shock, osmotic stress, and nutrient starvation consequently induce dramatic changes in the molecular composition of cellular machinery. Importantly, the cell's adjustment to a new homeostasis is accomplished through a variety of mechanisms, but most predominantly through the regulation of gene expression which is normally thought to function through the interplay of specific transcriptional repressors and activators. Recent evidence suggests, however, that more novel forms of transcriptional regulation may exist, including the repositioning of specific genes to distinct subdomains within the nucleus and through global changes in genome organization. Moreover, it has been hypothesized that such changes may function to supplement more traditional models of transcriptional regulation. Thus, upon environmental stress, cells may dramatically modify global transcriptional programs through a reorganization of their genome, facilitating a more rapid response to an adverse environment. The budding yeast Saccharomyces cerevisiae represents a remarkable model for elucidating how the organization of the genome and the repositioning of specific genes may function to regulate gene expression. In yeast, certain cellular stresses are known to stimulate the repositioning of genes within the nucleus and alter the global organization of the genome. Despite considerable research into the dynamic nature of genome organization, however, many questions remain as to the true function of gene positioning and the mechanism(s) by which cells establish specific organizational conformations of the genome.

In the following dissertation, I first provide evidence on the function of repositioning a specific gene to the nuclear periphery by examining the consequences of improperly localizing the GAL locus of budding yeast. Next, I describe my attempts to discern the coordinated diffusion of co-regulated genes as well as my findings on the independent movement of loci positioned various distances apart on the same chromosome. Finally, I describe novel findings on the mechanism of chromatin mobility through experiments focused on understanding the biological determinants of intracellular diffusion. Together, my results suggest that chromatin mobility is, in part, actively driven, which may function to facilitate more rapid alterations in genome organization and thus more rapid regulation of gene expression.

I first set out to understand the function of repositioning specific genes to distinct locations within the nucleus. In yeast, the GAL locus, comprised of GAL1, GAL7, and GAL10 and necessary for the metabolism of galactose, re-localizes from a central position of the nucleus when repressed to the nuclear periphery when transcriptionally active. To understand the function of this specific repositioning, I took advantage of mutants in different macromolecular complexes, namely the nuclear pore complex and the SAGA transcriptional complex, which both fail to properly localize the GAL locus. I demonstrate that the GAL locus is negatively regulated at the nuclear periphery as both mutants, when compared to wild- type, displayed massively upregulated transcription of GAL1 upon activation and a distinct lag in repression. Importantly, the increased induction kinetics of GAL1 mRNA is mirrored by increased expression levels of GAL1 protein, suggesting that the over-expressed GAL1 mRNA is functional and that export of the mRNA is not perturbed despite the mislocalization of the GAL locus. In my model, the GAL locus is positioned at the nuclear periphery to facilitate more rapid transcriptional repression when cells encounter their preferred carbon source, glucose.

Co-regulated genes may localize to transcriptional complexes known as transcription factories, hypothesized to facilitate more rapid and efficient transcription through colocalization and sequestration of specific transcriptional machinery. Because the GAL locus is known to re-localize at or near nuclear pore complexes upon activation, which may provide a scaffold for such transcriptional machinery, I next sought to observe transcription-induced coordinated diffusion of the two GAL loci within diploid yeast. To do so, I utilized a microscope capable of tracking both uniquely-labeled loci simultaneously using a double-helical point spread function. Although I did not discern evidence of potential transcription factories, I did reveal insights into the flexibility of chromatin by analyzing intrachromosomal loci separated by known distances. Overall, my results suggest that coordinated mobility of distinct loci correlates directly with their distance apart in space, regardless if they are intra or interchromosomal.

Finally, I explored the mechanism of chromatin mobility and, thereby, the driving force behind the re-organization of the genome by comparing the diffusion rates of chromatin and other macromolecules in distinct conditions. I began my investigation by starving cells of glucose and determining the effect on macromolecular mobility. Interestingly, both chromatin and mRNPs (messenger ribonucleoprotein particles) display a massive confinement in their mobility in such conditions. The reduction in mobility cannot be explained by a decrease in ATP, but can be replicated through a reduction of intracellular pH. I then show that both glucose starvation and reduction of intracellular pH induce a decrease in cell volume, increasing molecular crowding and reducing macromolecular mobility. Furthermore, I demonstrate a dependence of chromatin mobility on the cytoskeleton as simultaneous treatment with actin and microtubule depolymerizers specifically confines chromatin mobility. Lastly, by uncoupling metabolism and macromolecular mobility I propose that the preservation of an optimal cell volume is signaled independently from the energy status of the cell and discuss implications and future directions of this work.

The faithful and proper segregation of the genome between dividing cells is of paramount importance to all organisms. In order to maintain the integrity of the genetic information during division, cells make use of an extremely complex and highly regulated set of processes known collectively as mitosis. A key aspect of mitosis is the generation and maintenance of the mitotic spindle, a large and complex microtubule based structure that is responsible for organizing and transporting the chromosomes to the daughter cells. The mitotic spindle is of vital importance to the life of the cell, and multiple partially redundant pathways have evolved to regulate its assembly and operation. The small GTPase Ran governs one such pathway functioning in the vicinity of the chromosomes to control the activation of a variety of proteins that contribute to mitotic spindle assembly. However, in addition to promoting spindle assembly, the Ran pathway also regulates a number of other essential processes during the cell cycle such as nucleocytoplasmic transport and nuclear envelope dynamics. Due to this fact, studying the mitotic roles of the Ran pathway in vivo is challenging, as mitosis comprises only a small portion of the cell cycle. In order to overcome this obstacle, we took a small molecule inhibitor based approach, and sought to identify a compound capable of disrupting Ran pathway function with great temporal precision in living cells. In the following dissertation, we first provide an introduction to the cellular processes regulated by mitotic Ran pathway function, and then follow with descriptions of our efforts to develop and improve an inhibitor of RanGTP/importin–β function. Finally, we describe how we made use of this inhibitor to gain insight into a newly discovered role for Ran in the regulation of mitotic spindle positioning.

First we set out to develop a small molecule inhibitor capable of disrupting Ran pathway function. During interphase, the transport receptor importin–β carries cargoes into the nucleus, where RanGTP releases them. A similar mechanism operates in mitosis to generate a gradient of active spindle assembly factors around mitotic chromosomes. We implemented a FRET–based, high–throughput small molecule screen for compounds that interfere with the interaction between RanGTP and importin–β and identified importazole, a 2,4–diaminoquinazoline. We found that importazole specifically blocked importin–β–mediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1–mediated nuclear export. When added during mitosis, importazole impaired the release of an importin–β cargo FRET probe and caused both predicted and novel defects in spindle assembly. Together, our results identified importazole as a compound suitable for study of the Ran pathway in mitosis that specifically inhibits importin–β function, and suggest a possible molecular mechanism for importazole in which it alters importin–β interaction with RanGTP.

With an inhibitor of the pathway in hand, we attempted to improve importazole as a tool for study of mitotic Ran pathway function by elucidating its mechanism of action and devloping more potent analogues to maximize compound specificity. In order to gain further insight into importazole's molecular mechanism, we made use of surface plasmon resonance to directly measure the in vitro association between RanGTP and importin–β in the presence of importazole. In concordance with our previous observations, these experiments suggested that importazole does not destabilize the RanGTP/importin–β complex. However, the data was ultimately not reproducible enough to provide additional information about importazole function. In an effort to produce more potent inhibitors of RanGTP/importin–β function, we developed small molecule analogues based on the structure of importazole. One of these second generation compounds was capable of disrupting nucleocytoplasmic transport and mitotic spindle assembly, though it was not shown to be a significantly more potent inhibitor than importazole. Thus, we determined that importazole remains the best currently available tool for study of the Ran pathway in mitosis.

Finally, we took advantage of importazole to explore RanGTP/importin–β involvement in regulating mitotic spindle positioning, a mitotic function of the Ran pathway that has only recently been discovered. Proper positioning of the spindle is required to ensure correct segregation of the chromosomes during mitosis, and is mediated through pulling forces exerted on the astral microtubules by dynein/dynactin complexes linked to the cell cortex with Gαi, LGN, and the importin–β cargo protein NuMA. We found that importazole treatment disrupted mitotic spindle positioning in living cells without preventing formation of astral microtubules, and that it affected the cortical localization of both LGN and NuMA. These results demonstrated a role for RanGTP/importin–β function in spindle positioning, and our data suggest a model in which Ran may control this process through regulation of the stability of cortical positioning factors. A great deal remains to be learned about the role of the Ran pathway in mitotic spindle positioning, but importazole provides a promising avenue of study for this and other Ran mediated cellular processes.