The Evolution and Mechanics of Translational Control in Plants

The expression of numerous plant mRNAs is attenuated by RNA sequence elements located in the 5\u27 and 3\u27 untranslated regions (UTRs). For example, in plants and many higher eukaryotes, roughly 35% of genes encode mRNAs that contain one or more upstream open reading frames (uORFs) in the 5\u27 UTR. For this dissertation I have analyzed the pattern of conservation of such mRNA sequence elements. In the first set of studies, I have taken a comparative transcriptomics approach to address which RNA sequence elements are conserved between various families of angiosperm plants. Such conservation indicates an element\u27s fundamental importance to plant biology, points to pathways for which it is most vital, and suggests the mechanism by which it acts. Conserved motifs were detected in 3% of genes. These include di-purine repeat motifs, uORF-associated motifs, putative binding sites for PUMILIO-like RNA binding proteins, small RNA targets, and a wide range of other sequence motifs. Due to the scanning process that precedes translation initiation, uORFs are often translated, thereby repressing initiation at the an mRNA\u27s main ORF. As one might predict, I found a clear bias against the AUG start codon within the 5\u27 untranslated region (5\u27 UTR) among all plants examined. Further supporting this finding, comparative analysis indicates that, for ~42% of genes, AUGs and their resultant uORFs reduce carrier fitness. Interestingly, for at least 5% of genes, uORFs are not only tolerated, but enriched. The remaining uORFs appear to be neutral. Because of their tangible impact on plant biology, it is critical to differentiate how uORFs affect translation and how, in many cases, their inhibitory effects are neutralized. In pursuit of this aim, I developed a computational model of the initiation process that uses five parameters to account for uORF presence. In vivo translation efficiency data from uORF-containing reporter constructs were used to estimate the model\u27s parameters in wild type Arabidopsis. In addition, the model was applied to identify salient defects associated with a mutation in the subunit h of eukaryotic initiation factor 3 (eIF3h). The model indicates that eIF3h, by supporting re-initation during uORF elongation, facilitates uORF tolerance

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes.

BackgroundGeneral translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.ResultsHere, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.ConclusionsOur work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell

Translational Regulation in Arabidopsis thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3

Molecular functions of eukaryotic initiation factor 3 (eIF3) in translation are manifold, encompassing events from initiation complex assembly to translation termination. The contribution of the individual subunits of eIF3 to its multiple activities is quite unclear. It has been hypothesized that several of its 13 subunits contribute to mRNA specific regulation. Prior research had established that the h subunit of eIF3 in Arabidopsis was required for translation of specific mRNAs as well as for organ formation and meristem development. This study aims towards understanding the functions of individual subunits of eIF3 in the context of plant development and to further define the role of eIF3h at the molecular level. This dissertation describes an effort to identify mutations affecting each of the 13 eIF3 subunits. Using a panel of pollen-specific fluorescent marker genes, eIF3 subunits e, h and i1 were demonstrated to be essential for normal male gametophyte development. Furthermore, subunits b and c proved to be essential for embryo development. In contrast, a mutation in eIF3k revealed no phenotypic abnormalities. This work represents a systematic effort to attribute functions to many of the eIF3 subunits in growth and development in a multicellular eukaryote. The h subunit of eIF3 is necessary for the efficient translation of specific mRNAs in Arabidopsis. In particular, eIF3h fosters the translation of those mRNAs that harbor multiple upstream open reading frames (uORFs) in their 5’ leader. The specific molecular activity of eIF3h was investigated by structure-function analysis of the 5\u27 leader of the Arabidopsis AtbZip11 mRNA, which harbors a set of four uORFs that is evolutionarily conserved. By pairing extensive mutagenesis of the AtbZip11 5\u27 leader with gene expression analysis in Arabidopsis seedlings, it was revealed that eIF3h helps the ribosome to retain its reinitiation competence during uORF translation. These data establish a function for the h subunit of eIF3 in a special case of translation initiation, reinitiation. Finally, the molecular events during translation reinitiation were investigated further for a functional cooperation between eIF3h and the large subunit of the ribosome, given that the large ribosomal subunit had been implicated in reinitiation in other biological contexts. Reinitiation profiling using the AtbZip11 leader demonstrated that a protein of the large ribosomal subunit, RPL24B, bolsters reinitiation similar to eIF3h. Taken together, there exists a functional cooperation between the large ribosomal subunit and eIF3 that helps ribosomes to reinitiate after translation of uORFs

Genetic compartmentalization in the complex plastid of Amphidinium carteraeand. The endomembrane system (ES) in Phaeodactylum tricornutum

Peridinin-containing dinoflagellates are important members of single-celled eukaryotic algae, which arose from an engulfment of an ancient red alga by a so far undefined host cell, a process called secondary endosymbiosis. Their plastids feature a unique membrane architecture and are surrounded by only three membranes. As the reduction of the endosymbiont’s genome and gene transfer from the plastid to the nucleus, the whole plastid genome was reorganized into minicircles coding for genes normally coded on the plastid genome. In order to isolate individual minicircles from one representative peridinin-containing dinoflagellate Amphidinium carterae CCAM0512 a novel transposon-based approach was carried out within this thesis. 89 minicircles were therefore isolated from A. carterae, 18 (20.2 %) are gene-containing minicircles, 71 (79.8 %) are empty minicircles. The 18 gene-containing minicircles are divided into three groups of minicircles, six single-gene minicircles, one two-genes minicircle and one three-genes minicircle. The 71 empty minicircles are divided into six groups. The characteristics of these minicircles and unique features were analyzed in this thesis. In contrast to previously reported organellar RNA editing in peridinin-containing dinoflagellates, no RNA editing was observed on transcripts of minicircles of A. carterae based on the analysis of coding genes. Additionally, the transcription of open reading frames was shown in so-called empty minicircles. Finally, based on the comparison with minicircles and rDNA sequences of three other A. carterae strains, it was speculated that minicircles undergo a rapid evolutionary diversification. The mechanisms of protein (e.g. vacuolar proteins) transport and sorting have been well-studied in plants, yeast and animals. However, little is known in the diatom P. tricornutum. In order to investigate the protein transport and sorting in P. tricornutum, essential marker proteins have to be established. In this work, the identification of marker proteins in the endomembrane system was based on a combination of in silico search for homologous proteins of P. tricornutum to proteins with known localizations in plants and subsequent in vivo localization studies in P. tricornutum. Several markers for different subcellular compartments were identified including the plasma membrane, two vacuolar-like structures, cER, hER, the nuclear envelope and the second outermost membrane of the complex plastid (PPM). Furthermore, the three parts of the Golgi apparatus and the cytosol could also be marked. These useful subcellular marker proteins are a very important prerequisite for studying the mechanisms of protein transport and sorting in P. tricornutum

Molecular characterisation of the Polaris locus of Arabidopsis

This study is concerned with the analysis of the AtEMl0l promoter trap line of Arabidopsis thaliana. AtEMl0l seedlings show GUS expression in the tips of both primary and lateral roots, and more weakly in the hypocotyl and cotyledons. GUS activity in mature plants is found variably in both rosette and cauline leaves, stem nodes and also siliques but not other floral organs. Active auxins rapidly upregulate whilst cytokinins downregulate GUS transcript levels. AtEMl0l roots are shorter than those of the wild-type, a phenotype which is putatively linked to elevated ethylene levels. AtEMl0l roots were also found to be hypersensitive to exogenous cytokinins. Root patterning is not affected, but cells distal to the elongation zone are shorter in the AtEMl0l line than the wild-type. The T-DNA in line AtEMl0l was found to have inserted in a small, low abundance gene named POLARIS, which encodes a putative 36 amino acid polypeptide, which does not share homology to any known genes. POLARIS shows unusual genome organisation, with its 5' end overlapping with the 3' end of an upstream gene. Upstream sequence, embedded within the upstream gene, when fused to GUS were able to direct expression in root tips whilst a longer fragment mimics the GUS expression of the AtEMl0l line. Retransformation of the AtEMl0l line with a wild-type allele of POLARIS was able to complement the mutant phenotype indicating that the T-DNA insertion into POLARIS is responsible for the AtEMlOl phenotype. Overexpression of POLRIS resulted in transgenic plants with reduced sensitivity to both cytokinins and ACC. The structure of the POLARIS locus and the potential role of POLARIS in regulating cytokinin-induced ethylene levels, with regards to the control of root growth, are discussed

Study of GCN2 in Arabidopsis thaliana.

Li, Man Wah.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 109-119).Abstracts in English and Chinese.Thesis Committee --- p.IStatement --- p.IIAbstract --- p.III摘要 --- p.VAcknowledgements --- p.VIAbbreviations --- p.VIIIAbbreviations of Chemicals --- p.XList of Tables --- p.XIList of Figures --- p.XIITable of Contents --- p.XIIIChapter Chapter 1 --- Literature Review --- p.1Chapter 1.1 --- General amino acid control in yeast --- p.1Chapter 1.2 --- Mammalian eIF2α kinases --- p.7Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11Chapter 1.3 --- Plant General Amino Acid Control --- p.12Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14Chapter 1.4 --- Previous works in our lab --- p.15Chapter 1.5 --- Hypothesis and Objectives --- p.17Chapter Chapter 2 --- Materials and MethodsChapter 2.1 --- Materials --- p.18Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18Chapter 2.1.2 --- Primers --- p.21Chapter 2.1.3 --- Commercial kits --- p.25Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25Chapter 2.1.6 --- Enzymes --- p.25Chapter 2.1.7 --- Antibodies --- p.25Chapter 2.1.8 --- Equipments and facilities --- p.25Chapter 2.2 --- Methods --- p.26Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26Chapter 2.2.2 --- Basic molecular techniques --- p.27Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27Chapter 2.2.2.3 --- Restriction digestion --- p.27Chapter 2.2.2.4 --- DNA purification --- p.28Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28Chapter 2.2.2.6 --- DNA ligation --- p.29Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30Chapter 2.2.2.10 --- DNA sequencing --- p.30Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31Chapter 2.2.2.13 --- cDNA synthesis --- p.32Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33Chapter 2.2.2.15 --- Western blotting --- p.33Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47Chapter 2.2.6 --- Gel mobility shift assay --- p.47Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50Chapter 2.2.8.2 --- In vitro translation --- p.51Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53Chapter Chapter 3 --- ResultsChapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83Chapter Chapter 4 --- Discussion --- p.85Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97AppendicesAppendix I Commercial kits used in this project --- p.98"Appendix II Buffer, solution, gel and medium" --- p.99"Appendix III Chemicals, reagents and consumables" --- p.102Appendix IV Enzymes --- p.103Appendix V Antibodies --- p.104Appendix VI Equipments and facilities --- p.105Appendix VII Supplementary Data --- p.106Appendix VIII Amplification efficiency of real time primers --- p.108References --- p.10

Arabidopsis AMP-activated protein kinases in proteasomal complexes and their role in cell signaling

AMP-activated protein kinases (AMPK) are structurally and functionally highly conserved in all eukaryotes. The most profoundly studied yeast AMPK ortholog, Snf1, plays a role in the sensing and response to low energy and nutrient levels. There is much less information available about the function of plant Snf1 orthologs, termed Snf1-related protein kinases (SnRKs) and their regulatory roles in plant specific nutrient regulated mechanism, such as photosynthesis, plant hormone signaling and metabolism. Genetic studies of plant SnRK1 kinases are impeded by the lack of T-DNA insertion mutants in the SnRK1 catalytic subunits and the lack of male transmission of mutations affecting the SnRKγ subunit. The present work describes the isolation of T-DNA insertion mutations in SnRK1 β subunit genes and characterization of these mutants. Single and double mutant plants did not show any observable developmental or sugar signaling phenotype. Triple SnRK1β mutant could not be generated because no mutant allele is available for the third beta subunit. In our experiments overexpression of GFP-tagged SnRK1 beta subunits did not show subcellular relocalization in response to nutritional stress, a mechanism which was described in yeast. Instead, rapid proteasomal degradation of Arabidopsis SnRK1 subunits was observed in response to sugars and light. Proteasomal degradation of SnRK1 subunits prevented their efficient overexpression for biochemical studies. Former in vitro and in vivo studies found SnRK1 kinases in proteasomal complexes in Arabidopsis. This observation correlates with the fact that numerous nutrient status regulated metabolic enzymes are proteasomal substrates in Arabidopsis as in yeast and their degradation is controlled by phosphorylation and sugar availability. In order to identify SnRK1 interacting partners in vivo, it was attempted to isolate protein complexes from Arabidopsis through tagged SnRK1 subunits. In contrast to a former and mostly unsuccessful immunoaffinity purification approach, this work applies a rapid tandem affinity chromatography-based protein purification method. In addition to developing a biochemical approach for identification of SnRK1 interacting partners, we further explored the proteasomal connection of SnRK1 kinases and searched for their potential substrates using in vitro phosphorylation studies. Several sugar-regulated proteasomal substrates and F-box proteins were overexpressed in E. coli and purified to near homogeneity along with similar purification of SnRK1 kinase catalytic subunits. The purified proteins were subjected to in vitro kinase reactions using radioactive γ32P-ATP and phosphorylation was detected in more cases. Some of the phosphorylation sites were successfully identified by mass spectrometric analysis. Site-directed mutagenesis of the phosphorylation site on IAA6 and overexpression of the mutant construct indicates the importance of phosphorylation site in vivo. In order to provide further evidence for interaction of SnRK1 kinases with their substrates in vivo, an assay system for testing the stability of kinase substrates was developed