Translation and mRNA degradation are tightly regulated upon stress where protein synthesis and mRNA decay are modulated to optimize the stress response. However, the mechanisms that regulate mRNA decay and translation during stress are not fully understood. In this thesis, I show that Dcp2, a major decapping enzyme, undergoes phosphorylation by Ste20 kinase during stress and promotes stabilization of ribosomal protein mRNAs as well as Dcp2 accumulation in Processing bodies (P-bodies) in Saccharomyces cerevisiae. In addition, I have analyzed the role of P-bodies by examining how alterations in P-body assembly factors affect the transcriptome. Interestingly, I observe that Edc3, a component of P-bodies that promotes their assembly, can either stabilize or destabilize specific subsets of yeast mRNAs. I also show that Lsm4, a P-body component that mediates the assembly of P-bodies along with Edc3, promotes mRNA decay via its aggregation domain. These results argue that P-bodies can function as sites of mRNA degradation and storage for a subset of mRNAs by the localized accumulation of specific factors.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

One lesser-known but universal post transcriptional modification carried out in yeast and higher eukaryotes is the methylation of mRNA, as mediated by the Ime4 protein and its orthologs. Ime4 protein is essential for sporulation in yeast cells and for viability of higher eukaryotic cells. The precise locations of the Ime4 protein and the functions of the methylated mRNA are still largely unknown. Whereas Ime4 protein is believed to be exclusively nuclear in higher eukaryotes, we have observed the yeast Ime4 protein in the nucleus, in the cytosol and within cytosolic particles. These observations suggest that Ime4 could be a shuttling RNA binding protein, playing roles in the cytosol as well as the nucleus. As a first step to examining this idea, we tested the hypothesis that the punctuate cytosolic particles formed by Ime4 are P bodies. P bodies are transient aggregates of proteins and RNAs that form as a result of stresses such as glucose deprivation. This experiment was carried out using fluorescence microscopy using Ime4 tagged with GFP (green fluorescent protein) and the known P -body proteins Edc3, tagged with mCherry. We expected that if the proteins thus produced localized in the same place in the yeast cell, we could then deduce that Ime4 is present in P-bodies. We observed that Ime4 and Edc3 did not colocalize in the majority of cells, and thus concluded that the Ime4 granules are not P-bodies. However, our experiments showed instances of Ime4 signals near or around the P-bodies in some cells. Hence, the Ime4-containing aggregates are not likely to be P-bodies but could rather represent a different type of granule.

In the past few years, cytoplasmic processing bodies (P-Bodies) have been identified in eukaryotic cells. P-bodies have roles in translational repression, mRNA storage, mRNA decay and are conserved cytoplasmic aggregations of non-translating mRNAs in conjunction with translation repression and mRNA degradation factors. In this work, I, in collaboration with others provide evidence for a new biological role for P-bodies in viral life cycles. This work can be summarized thus:In a collaborative effort, I have identified connections between retrovirallike transposon life cycles and P-bodies. For example, genetic evidence in yeast indicates that key proteins within P-bodies are required for the life cycles of the Ty1 and Ty3 retrotransposons. Moreover, Ty3 genomic RNA (gRNA) as well as viral structural proteins accumulate in P-bodies, suggesting that P-bodies may serve as sites of viral assembly.Second, I have shown, with assistance of collaborators, that the positivestrand RNA virus, Brome Mosaic Virus (BMV) gRNA accumulates in P-bodies Moreover, viral RNA dependent RNA polymerase (RdRp) colocalizes with and co-immunoprecipitates with the P-body protein Lsm1p, suggesting that P-bodies may participate in viral replication. Remarkably, the accumulation BMV gRNA in P-bodies is dependent on cis-elements that have been demonstrated to play critical roles in viral RNA replication.The identification of P-bodies as sites of accumulation of viral gRNA and viral proteins of both retro-virus like elements and positive-stranded RNA viruses, expands the list of important biological roles played by P-bodies. Since P-body proteins and structure are highly conserved, these findings imply that Pbodies will be important for other RNA viruses.

No description available.

No description available.

Messenger RNA degradation is crucial for the regulation of eukaryotic gene expression. It not only modulates the basal mRNA levels but also functions as a quality control system, thereby controlling the availability of mRNA for protein synthesis. In Saccharomyces cerevisiae, the first and the rate-limiting step in the process of mRNA degradation is the shortening of the poly(A) tail by deadenylation complex. After the poly(A) tail shortens, mRNA can be degraded either through the major 5' to 3' decapping dependent or the 3' to 5' exosome-mediated degradation pathway. In this thesis, we show some of the means by which mRNA decay factors can modulate gene expression. First, Pat1 is a major cytoplasmic mRNA decay factor that can enter the nucleus and nucleo-cytoplasmically shuttle.  Recent evidence suggested several possible nuclear roles for Pat1. We analyzed them and showed that Pat1 might not function in pre-mRNA decay or pre-mRNA splicing, but it is required for normal rRNA processing and transcriptional elongation. We show that the mRNA levels of the genes related to ribosome biogenesis are dysregulated in the strain lacking Pat1, a possible cause of the defective pre-rRNA processing. In conclusion, we theorize that Pat1 might regulate gene expression both at the level of transcription and mRNA decay. Second, Edc3 and Lsm4 are mRNA decapping activators and mRNA decay factors that function in the assembly of RNA granules termed P bodies. Mutations in mRNA degradation factors stabilize mRNA genome-wide or stabilize individual mRNAs. We demonstrated that paradoxically, deletion of Edc3 together with the glutamine/asparagine-rich domain of Lsm4 led to a decrease in mRNA stability. We believe that the decapping activator Edc3 and the glutamine/asparagine-rich domain of Lsm4 functions together, to modify mRNA decay pathway by altering cellular mRNA decay protein abundance or changing the mRNP composition or by regulating P bodies, to enhance mRNA stability. Finally, mRNA decay was recently suggested to occur on translating ribosomes or within P bodies. We showed that mRNA degradation factors associate with large structures in sucrose density gradients and this association is resistant to salt and sensitive to detergent. In flotation assay, mRNA decay factors had buoyancy consistent with membrane association, and this association is independent of stress, translation, P body formation or RNA. We believe that such localization of mRNA degradation to membranes may have important implications in gene expression. In conclusion, this thesis adds to the increasing evidence of the importance of the mRNA degradation factors in the gene expression.

A recurring theme in biology is the aggregation of mRNA-protein complexes (mRNPs) into higher order assemblies. Often these complexes play important roles in the regulation of gene expression, but the function of the conserved cytoplasmic mRNP assemblies - P bodies and stress granules, is not known. It is believed that the misregulation of granule assembly is related to disorders like Amyotrophic Lateral Sclerosis and Frontotemporal Lobe Degeneration. Determining the complete composition of these granules may hold the key to understanding the function and mechanism of assembly of these granules. This work describes multiple approaches taken to identify new protein and mRNA components of P bodies and stress granules. New members of the P body and stress granule proteome reveal a role for these granules in diverse cellular processes including signal transduction, transcription and metabolism. Additionally, a new stress granule resident complex - the CCT complex, was also identified as a novel regulator of granule disassembly. This work also describes the first purification scheme for stress granules and presents a new system for in vitro study of stress granules. Together, the findings shed new light on the composition, function, structure and regulation of P bodies and stress granules in yeast.

Generally Hepatitis C Virus tropism is limited to hepatocytes. This limited tropism is a result of the receptors HCV requires for cellular entry and other host cellular factors including, uniquely, a liver specific miRNA, miR-122. The relationship between HCV and miR-122 is interesting, as commonly, miRNA are associated with suppression of function, but in the case of HCV, miR-122 actively promotes HCV proliferation. In-depth studies have demonstrated that miR-122 along with the RNA induced silencing complex (RISC) protein Argonaute 2 (Ago2) binds directly to two seed sequences separated by 8-9 nucleotides on HCV 5’UTR. Binding to the 5’UTR results in an increase in viral replication and translation. The method by which miR-122 promotes HCV translation and replication is not fully understood but evidence suggests that part of the function of miR-122 is to stabilize the HCV genome and protect it from exonuclease degradation by Xrn1, but other mechanisms remain to be identified. The reliance of HCV on miR-122 is best exemplified by the fact that removal of miR-122 by a miR-122 antagonist drastically impedes HCV viral titers in Chimpanzees and humans with no indication of escape mutants. The observation that HCV augmentation of the HCV life cycle by miR-122 requires Ago2 suggests that other components downstream in the miRNA suppression pathway may also be part of the mechanism of action. Our studies focused specifically on the processing body (p-body) associated DEAD-box helicase DDX6. DDX6 is essential for p-body assembly, required for robust miRNA suppression activity and elevated in HCV associated hepatocellular carcinomas. As such we hypothesized that DDX6 and p-bodies were directly or in-directly associated with the mechanism of action of miR-122. Knocking down DDX6 with siRNA indicated that DDX6 augments both HCV replication and translation. To examine whether DDX6 augmentation of HCV replication was related to the effects of miR-122 on the HCV life cycle, HCV replication and translation were assessed in the presence or absence of miR-122 when DDX6 was knocked down. Our data indicated that HCV replication and translation were augmented equally by miR-122 whether DDX6 was present or not. Our data also demonstrated that HCV replication and translation that was occurring independent of miR-122 was also still affected by DDX6 knockdown. Taken together our observations strongly suggest that the role DDX6 has on HCV is independent of HCV and miR-122’s relationship. In order to better understand miR-122’s relationship with HCV, we hypothesized that targeting the miR-122 binding region with siRNA would inhibit HCV replication initially, but that over the course of several rounds of treatment with the same siRNA, HCV would mutate to escape the siRNA, producing escape mutants that replicate without a dependency on miR-122. These escape mutants could be evaluated on how they replicate without using miR-122, shedding light on miR-122 and HCV’s relationship. Conversely if no escape mutants arose the siRNA could be further studied as a potential therapeutic for HCV. siRNA designed to target the miR-122 binding region inhibited HCV replication, confirming that the designed siRNAs could access the miR-122 binding region and function as an siRNA. Interestingly, when the siRNAs were used against a replication competent HCV RNA having a single nucleotide mutation in the first miR-122 binding site, instead of abolishing siRNA knockdown, two of the siRNA showed enhanced inhibition activity. The target sequences of these siRNAs spanned both miR-122 binding sites and we speculate that their inhibitory activity was due to competition for miR-122 binding to site 2. This observation indicates that siRNA targeting the miR-122 binding region have dual activity, by siRNA induced cleavage, and as a competitive inhibitor of miR-122 binding. Selection for viral escape mutants of the miR-122-binding site targeting siRNAs revealed viral RNAs having mutations within the miR-122 binding sites, in the surrounding region, and to other areas within the HCV IRES. The mutant viruses will be used to assess the influence of miR-122 binding site mutations on HCV replicative fitness, and to determine if the virus can evolve to replicate independent from augmentation by miR-122.