As cells age they develop distinctive characteristics that are highly associated with diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One of those highly associated characteristics is the formation of toxic protein aggregates like transactive response DNA binding protein (TDP43) that lead to disease pathology. To study the effects of TDP43 expression in aging cells we used growth assays, which showed that increased TDP43 expression is toxic to yeast cells. Our microscopy of aging cells expressing TDP43 also revealed that as cells age TDP43 expression becomes toxic, and nuclear expression seems to decrease as TDP43 expression moves to the cytoplasm and forms pathological aggregates. Due to the many important roles of TDP43 in the nucleus in its stable form, we hypothesized that in young cells low TDP43 expression wouldn’t be toxic but would become toxic as the cells aged. We found that at low expression levels of TDP43 we were able to maximize the amount of time at which we could image the aging cells before they died from the toxicity. This poses a possible mechanism by which we can observe important TDP43 expression characteristics for the entirety of the cell’s lifespan.

Regulating gene expression is crucial for the survival of yeast under fluctuating environmental conditions. Various regulating proteins and pathways are found to play important roles in responding to the environmental change at different levels. By utilizing two newly developed techniques: RNA sequencing and ribosome profiling, we are able to measure mRNA density and ribosome position of specific genes. Dom34, which has similar structure with eRF1, is known to dissemble stalled ribosome in mRNA, especially ribosomes arrested in the 3’UTR. In this thesis, we are focusing on how Dom34, regulate the presence of ribosomes in 3’UTR under different conditions. We verify potential Dom34 target genes and shows that Dom34 is partially inactivated under glucose starvation. Besides, we find no significant relation between translation recovery speed and ribosome distribution in 3’UTR. Though most starved genes will quickly return to their normal expression level after glucose re-addition, certain genes whose expression will be up-regulated under glucose deprivation remain in high level.

Translational elongation plays crucial role in mediating the expression of proteins and finally affecting various biological reactions and processes in living organisms. Reversely, the efficiency and rate of translational elongation is commonly regulated by amounts of internal elements, such as special codon or sequences, and external conditions, such as different nutrient conditions. The relevant effects have been well investigated a lot based on transcriptome or proteome analysis and ribosome profiling. Specifically, previous researches commonly identified lessening of protein production and ribosomal loading, which suggest the variation of elongation efficiency or ribosome stalling derived from poor genetic elements or worse nutrient conditions. We have found that, the existence of poly-proline sequence in nuclear-encoded mitochondrial proteins, such as ATP3 and Tim50, shows negative effects in the translation efficiency and the final products, while positive effects in mRNA localization on mitochondria. Since it is well-established that consecutive prolines will result in ribosomal stalling on mRNA, we assumed that the “stalling” may decrease the overall elongation rate, which can be measured and quantified in vivo. In addition, we also intend to monitor the translation variation of Yeast after switching from normal glucose condition to an unfamiliar condition, such different carbon source provide or even nutrient depletion. We finally realized that the translation of Yeasts is going to be impeded after alteration of nutrient supply, then great adaptation to the external “stress” is shown by organisms.

Protein production is tightly regulated by post transcription factors to control internal cellular elements and adapt themselves to the surrounding environment, such as the nutrient availability, temperature and oxygen absence. Translational elongation is one of the regulators for protein production. The mRNA localization to mitochondria during translational elongation has been reported to induce the increase in its protein production. The metabolic shift is a key factor for ATP synthase genes to localize its mRNAs to mitochondria during respiration to escalate ATP productions under oxidative phosphorylation. In the respiration, some ATP synthase genes are reported to induce the localization switching metabolism from fermentation to respiration. We have been investigating if slowing translational elongation can improve the protein production in conditionally localized mRNAs. Here we found that the ATP2 and ATP4 in yeast cells have decent increases in mRNA levels upon translational elongation slowing. In addition to it, the promoter has a certain effect in protein induction under respiration, but promoter combined with ORF has the stronger effect in it. To identify genes involved in mRNA localization-dependent gene expression, we have constructed the design of CRISPRi screening with a group of gRNAs targets various promoters in yeast cells in combination with a MCP- MS2 system to target a reporter mRNA to the mitochondria.

While we have learned that mRNA sequence strongly influences translation and co-translational pathways, the exact mechanisms by which this sequence and the RNA structures encoded impact these steps of gene expression are less understood. Therefore, more investigations are needed to reveal how translation elongation and efficiency is influenced by RNA sequence and how the affected translation regulates and determines a variety of co-translational pathways. In this dissertation, I explore the effects of translational kinetics on a series of co-translational pathways using the budding yeast Saccharomyces cerevisiae as a model organism. In Chapter 2, I developed an in-vivo elongation reporter in Saccharomyces cerevisiae to quantitatively monitor translation elongation duration and protein expression. Using this elongation reporter, I investigated the effects of elongation stalls induced by different types of genetic factors on gene expression and demonstrated that distinct ribosomal stalls may trigger distinct co-translational pathways. In Chapter 3, I studied co-translational mRNA localization to mitochondrion, and proposed that translational kinetics, such as ribosomal stall caused by polyprolines, play an important role in mediating co-translational import. In addition, I further studied the effects of elongation stalls on mitochondrial import stress and triggering of relevant quality control pathways. In Chapter 4, I investigated the mechanism of cytosolic mRNP granule formation under glucose deprivation condition. In this study, I use CRISPRi to knockdown expression of RVB2 and confirm its essential role in deciding the fate of mRNA localization and translatability after glucose depletion. Finally in Chapter 5, I address the enhancements made to the developed method, outline potential future directions for the research presented in this dissertation, and conclude with my final remarks.

When yeast cells endure environmental changes, they respond transcriptionally and translationally causing a fluctuation of gene expression dependent on the environmental changes. Understanding how yeast cells molecularly respond to environmental changes can give us insight into certain molecular mechanisms that contribute to cellular responses. We investigated how yeast cells molecularly respond during a 5-day growth period in which they will endure glucose deprivation causing metabolization of ethanol that produces a diauxic shift resulting in respiratory growth, before the cells reach stationary phase. During the growth period, yeast develop stress granules during respiratory growth. The functions of stress granules are not known but are indicated to have a role in gene regulation during environment changes. Ded1 is an RNA helicase important for translation initiation that is a contributing factor to the assembly and disassembly of stress granules by co-localizing to them. A recently discovery found deleting Ado1 completely disrupts co-localization of Ded1 to stress granules. Giving us the opportunity to use the disruption to understand how yeast cells endure the 5-day growth period but also attempt to discover a possible function for stress granules.

Protein translation is regulated at the stages of initiation, elongation, and termination. Under stress, translation is generally suppressed. Translation initiation has traditionally been thought of as the key control step of translation across a variety of stresses. Here we find that during glucose starvation in yeast, differential translation elongation is a crucial mechanism that allows preferential translation of select genes during stress. While these growth mRNAs have similar overall ribosome occupancy compared to these well-translated stress mRNAs, their distribution of ribosomes is skewed towards the 3’ end of the ORF. We also find that the skewed distribution in ribosomes during stress strongly correlates with increased mRNA binding by the DEAD-box RNA helicase, Dhh1. We speculate the recruitment of Dhh1 is associated with this differential protein translation upon glucose starvation. By regulating translation through elongation, cells could more dynamically control protein synthesis in adverse conditions.

Protein aggregation is a hallmark of aging and age-related diseases. Meanwhile, physiological aggregation of proteins and other bio-molecules is used by cells as a strategy to maintain cellular fitness under various conditions. RNA-binding proteins, possibly due to their intrinsically disordered and aggregation-prone nature, are associated with many pathological and physiological aggregates. Here we examine the subcellular localization of a group of proteins from two mRNP structures during yeast replicative aging by fluorescence microscopy. We find that several P-body components form an aggregate during aging in yeast. This P-body-like aggregate differs from normal yeast P-bodies in its insensitivity to glucose starvation and the absence of certain mRNA species. Similar to yeast P-bodies, it has a relatively dynamic structure, which can be disrupted by hexanediol treatment and upon the inhibition of protein translation. Furthermore, the aggregate does not contain protein disaggregase Hsp104, a marker for misfolded protein inclusions, under different conditions and thus unlikely to be a physiological deposition site of misfolded proteins. Combining several lines of evidence, we speculate that the age-related P-body-like aggregate represents an aberrant state of P-bodies with altered composition and function. We propose that fully understanding its formation and function in yeast will provide insights into mechanisms that underlie human aging and age-related diseases.

Mitochondria are organelles whose function, protein composition, size, and morphology are highly variable and regulated in response to nutrient availability and other environmental conditions. Their crucial role in metabolism involves the production of adenosine triphosphate (ATP), the energy currency of the cell, by the oxidative phosphorylation pathway. TEM studies observed ribosomes enriched at the mitochondrial surface, suggesting that mRNAs localize co-translationally. mRNA localization is a post transcriptional method for regulating gene expression in parallel with transcriptional methods. While mRNA localization is a way to control protein production or limit translation activity to specific cellular locations, the potential of mRNA localization as a strategy for altering the composition of mitochondrial proteins in different environmental conditions has not been explored. For brewer’s yeast, 99% of mitochondrial proteins are encoded in the nuclear genome. To ensure mitochondrial function, nuclear-encoded proteins are imported into the mitochondria through mitochondrial translocases on the outer mitochondrial membrane. mRNA localization is implicated in mitochondrial protein homeostasis along two axes: it helps synchronize the nuclear and mitochondrial genome translation programs to ensure proper stoichiometry of nuclear- and mitochondrial-encoded proteins and it is required for initiating the co-translational import of highly hydrophobic nascent peptides that are vulnerable to aggregation and misfolding in the cytosol. While it is clear that mRNA localization is important for mitochondrial biogenesis and homeostasis, the mechanism of localization has not been fully elucidated for mRNAs that do not have known RNA-binding proteins partners that regulate their localization. More than 200 genes associate to the mitochondria after translating a 5’ amphiphilic mitochondria targeting sequence (MTS) that can interact with translocation machinery only after the mRNA-ribosome complex has found the mitochondria through diffusive search. Additionally, the nascent peptides produced by co-translationally localized mRNAs bind to the chaperones Hsp70 (in brewer’s yeast) and Hsp90 (in mammalian cells) that are implicated in the proper recognition of the preprotein by other components of translocation machinery. Based on our understanding of the MTS-driven mechanism of mRNA localization, I developed a mathematical model and stochastic simulation of translation, peptide signal maturation, and mRNA diffusion in cells of varying mitochondrial volume. After reproducing experimental observations of mRNA localization in diverse cellular states, including in fermentative versus respiratory metabolic conditions, we predicted that increasing translation duration would drive mRNA localization in vivo. However, quantitative microscopy in brewer’s yeast reveals that ribosome stalls downstream of the MTS drive localization regardless of translation duration. This has given us new insights into the peptide signal maturation component of MTS-driven localization mechanism, and has been incorporated into the stochastic simulation as well as experimental design. The mechanism of mRNA localization is based on fundamental processes like translation and diffusion instead of relying on gene-specific or condition-specific regulatory factors. Therefore, we postulate that it is conserved to mammalian systems given the high degree of conservation of mammalian genes and peptide-binding chaperones. We find that our stochastic simulation of translation, peptide signal maturation, and mRNA diffusion can reproduce experimental observations of mRNA localization behaviors in brewer’s yeast and mammalian cells, indicating that the biophysical mechanism of MTS-driven localization is conserved between eukaryotes. The combination of translation and diffusion kinetics is a novel mechanism for regulating mitochondrial gene expression post-transcriptionally across eukaryotes and adds to our understanding of mitochondrial homeostasis and mitochondrial biogenesis in shifting environmental conditions.

Processing bodies (p-bodies) are transient, membrane-less constructs consisting of mRNA and RNA-binding proteins. Though constitutively expressed in mammalian cells, formation of these structures in yeast is induced by metabolic stress. While not all mRNAs localize to membrane-less granules, identifying their contents has proved difficult because of the structural nature of these droplets. It therefore becomes necessary to stabilize these granules to qualitatively observe mRNA localization. Here we present a new methodology for granule stabilization using a combination of UV cross-linking and the small dialdehyde Glyoxal. We also provide evidence suggesting Glyoxal to be an effective fixative for membrane less granule fixating in mammalian cells.