Browsing by Subject "Messenger RNA"
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Item Characterization of Arabidopsis thaliana translation initiation factor eIF4F(2006-08) Provinzino, Anne Marie Schneider; Browning, Karen S.Eukaryotic translation initiation is a complex process involving many factors. Two vital components of this system are eukaryotic initiation factors (eIF) 4E and 4G, which together form the complex eIF4F. eIF4E is the cap-binding subunit, interacting with the 7-methyl guanosine at the 5'-end of messenger RNA. There are multiple forms of this gene in Arabidopsis thaliana: eIF4E1, eIF4E2, eIF4E3, eIFiso4E, and novel cap-binding protein. The scaffolding component of the eIF4F cap-binding complex is eIF4G. This protein binds not only to eIF4E, but also to eIF3, eIF4A, and poly(A)binding protein. Three versions are found in Arabidopsis: eIF4G, eIFiso4G1, and eIFiso4G2. Plants are unique in that they are the only organisms to contain the isoform versions of these genes. A goal of the research described in this thesis is to determine the mechanisms by which plant eIF4F isoforms specifically select mRNAs for translation. This thesis describes the cloning of dicistronic Arabidopsis eIF4G with some members of the eIF4E cap-binding family. Comparison of the biochemical activities of these complexes with various mRNAs will generate more information about their specific functions. Analysis of Arabidopsis T-DNA insertion mutants is included to elucidate the role(s) of eIF4F, eIFiso4F, and their subunits in plant translation. The phenotype(s) of a mutant may provide clues as to the function of the protein(s) not expressed. The importance of the eIF4F complex to translation initiation makes it a target for regulation. These regulatory mechanisms include inhibition by cleavage, competitive inhibitors, phosphorylation, binding of partner proteins, and exploitation by viruses. Our hypothesis is that the eIF4E and eIF4G genes are also regulated at the level or translation. Part of this thesis describes preparation and sequencing of cDNAs for eIF4G, eIFiso4G1, eIFiso4G2, eIF4E1, eIF4E2, eIF4E3, eIFiso4E, and nCBP to identify full length 5'UTRs and determine if regulatory elements are present.Item Design Principles for Ligand-Sensing, Conformation-Switching Ribozymes(Public Library of Science, 2009-12-24) Chen, Xi; Ellington, Andrew D.Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications.Item Distinct roles for the 5' and 3' untranslated regions in the degradation and stability of chloroplast tufA mRNA(2005) Zicker, Alicia A.; Herrin, David L.The tufA gene encodes the chloroplast elongation factor Tu and is found in the chloroplast genome in Chlamydomonas reinhardtii, whereas it is in the nucleus in land plants. When C. reinhardtii is treated with chloramphenicol (CAP), an inhibitor of ribosome elongation, the stability of the 1.7-kb tufA mRNA is increased, and a RNA of ~1.5 kb over-accumulates. When CAP and the transcription inhibitor rifampicin are added together, tufA mRNA can be chased into the 1.5-kb RNA (which is subsequently degraded), suggesting that it is an intermediate in the degradation pathway. Mapping of the 5' and 3' ends of the 1.5-kb RNA showed that it is truncated at the 5' end, and is missing 177-181 nucleotides; there was no evidence of truncation at the 3' end. The 5' end of this intermediate lies 76-80 nucleotides upstream of the tufA start codon, and within a vii small ORF that has predicted secondary structure. Interestingly, the degradation intermediate did not over-accumulate in a chloroplast ribosome-deficient mutant, suggesting that its stabilization requires elongation-arrested ribosomes. Finally, evidence was obtained that the intermediate accumulates in the absence of CAP, especially in older cultures. These results indicate that a major pathway for degradation of tufA mRNA in vivo begins at the 5' end of the RNA, probably with an endonucleolytic cleavage, but 5' to 3' exonuclease activity cannot be ruled out. To determine whether the 5' or 3' UTR of tufA is destabilizing, the corresponding regions of an atpA-aadA-rbcL gene (which gives high mRNA levels) were replaced with one or both tufA UTRs, and transformed into the chloroplast. Northern blot analysis of the transformants indicated, quite surprisingly, that the 3' UTR of tufA is strongly destabilizing. The results suggest that the UTRs of tufA have distinct, but also interacting roles in mediating decay of tufA mRNA. To determine if the 3' UTR of tufA can regulate protein expression, constructs were made using Renilla luciferase as reporter, and transformed into chloroplasts. Luminescence measurements of the transformants indicated that the 3' UTR of tufA can regulate expression at the protein level, but no more so than the 5' UTRItem Functional characterization of the role of Bruno protein in translational regulation and germ line development in Drosophila melanogaster(2006-05) Yan, Nan, 1979-; Macdonald, Paul M.Both body axes of the Drosophila egg are determined by localization of several mRNAs to specific regions within the oocyte. One of these mRNAs, oskar (osk), is required for posterior body patterning. Localization and translational control are both crucial for the correct deployment of osk. Bruno (Bru) binds specifically to the 3’UTR of the osk mRNA and represses osk translation. In this dissertation, I first describe a genetic screen looking for dominant modifiers of the arrest (aret) mutant phenotype (aret encodes Bru). Two modifiers suggested additional targets for Bru action. One is Star, a gene that contributes to provision of Gurken activity. The second suggested target is a gene acting in the Delta signaling pathway. A final modifier, Lk6, encodes a protein kinase predicted to regulate eIF4E. I also took a biochemical approach trying to understand how Bru regulates osk translation. Bru protein contains three RNA Recognition Motifs, but the remainder of the protein had no known function. I identified a domain, which is required for interaction to Bru itself, Cup and Apontic. Subsequent analysis of mutant forms of Bruno defective in these interactions led us to an unexpected discovery that Bru also acts as an activator of osk translation. Parallel analysis of Bru binding sites in osk 3’UTR fully support the notion that Bru has a dual role. There are two clusters of Bru Recognition Elements in either end of osk 3’UTR. Point mutations in one cluster cause overproduction of Osk protein while point mutations in the other cluster largely prevent translation of the message. To understand the molecular basis of the opposing roles of Bru, I used quantitative methods to better define and compare the binding of Bru to the different regulatory elements: those that either repress or activate osk mRNA translation. Using purified components I found that Bru binds to two clusters of binding sites in the osk 3’UTR differently, in terms of affinity, cooperativity and apparent compaction of the RNA. This work raises the possibility that the details of how Bru binds its substrate may determine whether it acts as a repressor or an activator.Item Gene Expression Signatures of Energetic Acclimatisation in the Reef Building Coral Acropora millepora(Public Library of Science, 2013-05-09) Bay, Line K.; Guérécheau, Aurélie; Andreakis, Nikos; Ulstrup, Karin E.; Matz, Mikhail V.Background -- Understanding the mechanisms by which natural populations cope with environmental stress is paramount to predict their persistence in the face of escalating anthropogenic impacts. Reef-building corals are increasingly exposed to local and global stressors that alter nutritional status causing reduced fitness and mortality, however, these responses can vary considerably across species and populations. -- Methodology/Principal Findings -- We compare the expression of 22 coral host genes in individuals from an inshore and an offshore reef location using quantitative Reverse Transcription-PCR (qRT-PCR) over the course of 26 days following translocation into a shaded, filtered seawater environment. Declines in lipid content and PSII activity of the algal endosymbionts (Symbiodinium ITS-1 type C2) over the course of the experiment indicated that heterotrophic uptake and photosynthesis were limited, creating nutritional deprivation conditions. Regulation of coral host genes involved in metabolism, CO2 transport and oxidative stress could be detected already after five days, whereas PSII activity took twice as long to respond. Opposing expression trajectories of Tgl, which releases fatty acids from the triacylglycerol storage, and Dgat1, which catalyses the formation of triglycerides, indicate that the decline in lipid content can be attributed, at least in part, by mobilisation of triacylglycerol stores. Corals from the inshore location had initially higher lipid content and showed consistently elevated expression levels of two genes involved in metabolism (aldehyde dehydrogenase) and calcification (carbonic anhydrase). -- Conclusions/Significance -- Coral host gene expression adjusts rapidly upon change in nutritional conditions, and therefore can serve as an early signature of imminent coral stress. Consistent gene expression differences between populations indicate that corals acclimatize and/or adapt to local environments. Our results set the stage for analysis of these processes in natural coral populations, to better understand the responses of coral communities to global climate change and to develop more efficient management strategies.Item Genetic and biochemical studies on the differential modulation of RNA decay and processing by inhibitory proteins in Escherichia coli(2006) Zhao, Meng; Georgiou, GeorgeThe regulation of mRNA decay is a critical post-transcriptional step in the control of gene expression. In E. coli, RNase E carries out the first and rate-limiting step in the decay of most mRNAs, as well as in the processing of ribosomal, transfer RNAs and small regulatory RNAs. The RNase E protein has two domains: the catalytic N-terminal half and the C-terminal half containing the scaffold region for the assembly of an RNA degrading machine termed the degradosome. Earlier studies in our lab identified the trans-acting proteins, RraA and RraB, which inhibit RNase E activity through direct-interaction with the enzyme. The present work explores several mechanistic, physiological and biotechnology-related aspects of the modulation of E. coli RNA decay and processing by inhibitory proteins. We found that, in contrast to RraA, RraB interacts with a different site on RNase E, results in distinct changes in degradosome composition, and interferes with cleavage of a different set of transcripts. Therefore, our results revealed a novelItem Measuring system dynamics: mRNA, protein and metabolite profiling(2005) Lu, Peng; Marcotte, Edward M.Compared to the traditional reductionist approach, systems biology seeks to explain biological phenomenon, not on a gene-by-gene basis, but through the net interactions of all cellular and biochemical components within a cell or organism. As systems biology is driving technological developments, we sought to improve high-throughput measurements of the major cellular molecules and apply multiple molecular profiling approaches to measure cellular system dynamics. We focused on three classes of molecules: mRNA, proteins and metabolites. For mRNA, expression deconvolution, a new algorithm for expression pattern analysis, was proposed to reveal dynamic changes in cell populations by reinterpretation of DNA microarray data. For proteins, a novel statistical method was established to calculate protein expression levels from shotgun proteomics; protein levels measured by this approach correlate well with protein abundance measured by Western blot and 2D gels. For metabolites, we took advantage of the extended 13C NMR spectral range and developed 1 H-13C 2D-NMR for in vitro and in vivo metabolic profiling of cells. With these technologies, we combined mRNA, protein and metabolite profiling to study one carbon metabolism and the yeast cell cycle. Integrating various “omic” data, we showed that local changes in one carbon metabolism (AdoMet hyperaccumulation) causes a gross change in the global metabolome, accompanied by both transcriptional and post-transcriptional responses, ultimately leading to a G1-delay defect in the cell cycle. We began mapping the yeast cell cycle in terms of dynamic abundance changes of the major cellular molecules. All these studies indicate that for many cases the measurement of mRNA is not predictive of the corresponding protein or metabolite abundances. Consequently, these different types of data provide complementary information to elucidate control mechanisms otherwise evident. This validates an essential idea of systems biology: it is only by integrating different levels of biological information that the cell’s state can be fully described.Item miRNA-Dependent Translational Repression in the Drosophila Ovary(Public Library of Science, 2009-03-02) Reich, John; Snee, Mark J.; Macdonald, Paul M.Background: The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary. Methodology/Principal Findings: Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed. Conclusions/Significance: Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.Item MRNA degradation in the control of gene expression in yeast(2001-08) Brown, Justin Travis; Johnson, Arlen W.The pathways for eukaryotic mRNA translation and degradation are composed of numerous interconnected elements. I have characterized the general cellular roles of factors involved in mRNA degradation pathways and their part in the control of gene expression in the budding yeast Saccharomyces cerevisiae. Xrn1p is the non-essential cytoplasmic 5´ exoribonuclease required for rapid mRNA turnover. xrn1 mutants have been associated with wildly disparate phenotypes, including karyogamy and meiotic recombination. I identified two translation mutations (in eIF4E and eIF2B) from a synthetic lethal screen, supporting my assertion that aberrant gene expression contributes to xrn1 pleiotropy. I then demonstrated that mutations in capping enzyme and in eIF4G genetically interact with xrn1. My results contradict the currently held model that stabilization of mRNAs by deletion of XRN1 should suppress the inviability of upstream translation mutations. I conclude that the accumulation of messages in an xrn1 mutant is in fact lethal in combination with particular defects in translation. A previous xrn1 synthetic lethal screen identified SKI2 and SKI3. My screen also identified mutations complemented by SKI4/CSL4, SKI6/RRP41, RRP46, and SKI8. Debate persists over the primary role of the Ski proteins. PolyA-minus mRNA is not translated efficiently in wild-type eukaryotic cells, but is translated efficiently in ski mutants, perhaps due to altered translational specificity. However, as the SKI genes are required for 3´ mRNA degradation, it is possibly a consequence of inhibition of 3´ mRNA decay. I show that Ski2p, Ski3p and Ski8p form a stable complex and that Ski2p and Ski3p are cytoplasmic, not nuclear as previously maintained. To further distinguish between the Ski models and to directly assess the heretofore unexamined effects of 3´ mRNA degradation on translation in non-mutant cells, I show that an RNA can be translated efficiently in wild-type cells without polyA or Pab1p when 3´ degradation is blocked in cis. In addition, this enhanced expression phenocopies a ski mutant. Hence, functional redundancy is the simplest model to explain synthetic lethality between xrn1 and ski mutations: mRNA decay is an essential process and lethality consequently arises from inhibition of both 5´ and 3´ pathways for mRNA degradation.Item Regulation of elements of the thyroid hormone and corticosteroid systems by stress, hormone treatment, and atrazine during ontogeny of red drum (Sciaenops ocellatus)(2008-08) Applebaum, Scott Lee, 1974-; Nunez, B. ScottSurvival of teleost larvae requires growth and development which depend upon endocrine processes. In this dissertation I have examined the ontogeny of elements of the thyroid hormone (TH) and corticosteroid (CS) systems in red drum (Sciaenops ocellatus). Basal cortisol production was first detected 3 days post-hatch (DPH) and a cortisol stress response was present from 6 DPH forward. Changes in steroidogenic enzyme mRNA (CYP11B and CYP21) levels did not correlate with these events. The time necessary to reach peak cortisol levels as well as return to basal levels declined as larvae developed. A second set of studies examined ontogenetic patterns in levels of mRNAs encoding thyroid (soTR[alpha] and soTR[beta]) and corticosteroid (soGR) hormone receptors and assessed the regulation of these mRNAs by exogenous triiodothyronine (T3) and cortisol. soTR[alpha], soTR[beta] and soGR were expressed in all stages of red drum examined. soTR[alpha] levels increased during the time when surging TH levels have been reported. soTR[beta] levels did not differ significantly during development. soGR levels were strongly correlated with those of soTR[alpha]. T3 up-regulated soTR[alpha] and soTR[beta] levels in 7 DPH, but not older larvae. Cross-regulation of receptor mRNAs by exogenous treatment with T3 or cortisol was not observed. Finally, I assessed the influence of a common herbicide, atrazine, on receptor mRNA, TH levels and growth of red drum. In two experiments, red drum exposed to environmentally relevant levels of atrazine did not alter hormone receptor mRNA levels, or TH content. However, atrazine did depress growth in some instances. In conclusion, the expression patterns of hormone receptor mRNA in embryos suggests receptor proteins could be activated by maternal hormones prior to the onset of endogenous hormone production. A correlation between soTR[alpha] and soGR mRNA levels suggests coordinated function of TH and CS systems, although regulatory interactions between these systems were not evident under the conditions in this study. Patterns in soTR[alpha] and soTR[beta] mRNA levels support an important role for TH in the larval to juvenile transformation of red drum larvae. The results also support growing evidence indicating atrazine exposure effects larval growth and may impact their survival in the wild.Item Role of DksA and Hfq in Shigella flexneri virulence(2007-05) Sharma, Ashima Krishankumar, 1979-; Payne, Shelley M.Hfq is a post-transcriptional regulator playing an important role in virulence and cellular physiology by regulating the expression of several genes either directly or indirectly through interaction with small regulatory RNAs (sRNA). Hfq is highly abundant and its synthesis in E. coli is subject to auto-repression at the level of translation. My studies with Shigella flexneri showed that hfq gene expression is regulated at the transcriptional level by a pleiotropic regulatory protein, DksA. I compared the gene expression profiles of wild type and dksA mutant S. flexneri by microarray and real time PCR analyses and determined that hfq expression was reduced in the dksA mutant. Significantly reduced Hfq levels in the dksA mutant were restored to wild type levels in the dksA mutant complemented with wild type dksA. Characterization of an hfq mutant in S. flexneri showed several phenotypes in common with the dksA mutant including reduced ability to survive in stress conditions and formation of elongated cells within cultured epithelial cells. Because DksA is required by S. flexneri to form plaques in cultured epithelial cell monolayers, a measure of virulence, the role of Hfq in the dksA virulence phenotype was assessed. Inducing expression of hfq in the dksA mutant restored plaque formation, and an S. flexneri hfq mutant failed to form wild type plaques. These data suggest that DksA plays a role in regulating hfq gene expression and that this regulation is important for S. flexneri virulence. In an in vitro transcription assay, addition of purified DksA increased transcription of hfq and this effect was greater with one of the two known hfq promoters. Addition of ppGpp, a stringent response molecule, along with DksA in the in vitro transcription assay resulted in a further increase in transcription of hfq, indicating that DksA is required for maximal transcription of hfq during both exponential and stringent response growth conditions. Real time PCR analysis showed reduced mRNA levels of the three major transcriptional activators of S. flexneri virulence genes, VirF, VirB and MxiE in the hfq mutant providing an explanation for its reduced ability to invade and form plaques in cultured monolayers.Item Unstable Transcripts in Arabidopsis Allotetraploids Are Associated with Nonadditive Gene Expression in Response to Abiotic and Biotic Stresses(Public Library of Science, 2011-08-29) Kim, Eun-Deok; Chen, Z. JefferyGenome-wide analysis has documented differential gene expression between closely related species in plants and animals and nonadditive gene expression in hybrids and allopolyploids compared to the parents. In Arabidopsis, 15–43% of genes are expressed differently between the related species, Arabidopsis thaliana and Arabidopsis arenosa, the majority of which are nonadditively expressed (differently from mid-parent value) in allotetraploids. Nonadditive gene expression can be caused by transcriptional regulation through chromatin modifications, but the role of posttranscriptional regulation in nonadditive gene expression is largely unknown. Here we reported genome-wide analysis of mRNA decay in resynthesized Arabidopsis allotetraploids. Among ~26,000 annotated genes, over 1% of gene transcripts showed rapid decay with an estimated half-life of less than 60 minutes, and they are called allotetraploid genes with unstable transcripts (AlloGUTs). Remarkably, 30% of alloGUTs matched the nonadditively expressed genes, and their expression levels were negatively correlated with the decay rate. Compared to all genes, these nonadditively expressed alloGUTs were overrepresented 2-6-fold in the Gene Ontology (GOSlim) classifications in response to abiotic and biotic stresses, signal transduction, and transcription. Interestingly, the AlloGUTs include transcription factor genes that are highly inducible under stress conditions and circadian clock regulators that regulate growth in A. thaliana. These data suggest a role of mRNA stability in homoeologous gene expression in Arabidopsis allopolyploids. The enrichment of nonadditively expressed genes in stress-related pathways were commonly observed in Arabidopsis and other allopolyploids such as wheat and cotton, which may suggest a role for stress-mediated growth vigor in hybrids and allopolyploids.Item Virus-Encoded microRNAs: An Overview and a Look to the Future(Public Library of Science, 2012-12-20) Kincaid, Rodney P.; Sullivan, Christopher S.MicroRNAs (miRNAs) are small RNAs that play important roles in the regulation of gene expression. First described as posttranscriptional gene regulators in eukaryotic hosts, virus-encoded miRNAs were later uncovered. It is now apparent that diverse virus families, most with DNA genomes, but at least some with RNA genomes, encode miRNAs. While deciphering the functions of viral miRNAs has lagged behind their discovery, recent functional studies are bringing into focus these roles. Some of the best characterized viral miRNA functions include subtle roles in prolonging the longevity of infected cells, evading the immune response, and regulating the switch to lytic infection. Notably, all of these functions are particularly important during persistent infections. Furthermore, an emerging view of viral miRNAs suggests two distinct groups exist. In the first group, viral miRNAs mimic host miRNAs and take advantage of conserved networks of host miRNA target sites. In the larger second group, viral miRNAs do not share common target sites conserved for host miRNAs, and it remains unclear what fraction of these targeted transcripts are beneficial to the virus. Recent insights from multiple virus families have revealed new ways of interacting with the host miRNA machinery including noncanonical miRNA biogenesis and new mechanisms of posttranscriptional cis gene regulation. Exciting challenges await the field, including determining the most relevant miRNA targets and parlaying our current understanding of viral miRNAs into new therapeutic strategies. To accomplish these goals and to better grasp miRNA function, new in vivo models that recapitulate persistent infections associated with viral pathogens are required.