Transfer RNA modifications and genes for modifying enzymes in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>In all domains of life, transfer RNA (tRNA) molecules contain modified nucleosides. Modifications to tRNAs affect their coding capacity and influence codon-anticodon interactions. Nucleoside modification deficiencies have a diverse range of effects, from decreased virulence in bacteria, neural system disease in human, and gene expression and stress response changes in plants. The purpose of this study was to identify genes involved in tRNA modification in the model plant <it>Arabidopsis thaliana</it>, to understand the function of nucleoside modifications in plant growth and development.</p> <p>Results</p> <p>In this study, we established a method for analyzing modified nucleosides in tRNAs from the model plant species, <it>Arabidopsis thaliana </it>and hybrid aspen (<it>Populus tremula </it>× <it>tremuloides</it>). 21 modified nucleosides in tRNAs were identified in both species. To identify the genes responsible for the plant tRNA modifications, we performed global analysis of the Arabidopsis genome for candidate genes. Based on the conserved domains of homologs in <it>Sacccharomyces cerevisiae </it>and <it>Escherichia coli</it>, more than 90 genes were predicted to encode tRNA modifying enzymes in the Arabidopsis genome. Transcript accumulation patterns for the genes in Arabidopsis and the phylogenetic distribution of the genes among different plant species were investigated. Transcripts for the majority of the Arabidopsis candidate genes were found to be most abundant in rosette leaves and shoot apices. Whereas most of the tRNA modifying gene families identified in the Arabidopsis genome was found to be present in other plant species, there was a big variation in the number of genes present for each family.</p> <p>Through a loss of function mutagenesis study, we identified five tRNA modification genes (AtTRM10, AtTRM11, AtTRM82, AtKTI12 and AtELP1) responsible for four specific modified nucleosides (m¹G, m²G, m⁷G and ncm⁵U), respectively (two genes: AtKTI12 and AtELP1 identified for ncm⁵U modification). The <it>AtTRM11 </it>mutant exhibited an early-flowering phenotype, and the <it>AtELP1 </it>mutant had narrow leaves, reduced root growth, an aberrant silique shape and defects in the generation of secondary shoots.</p> <p>Conclusions</p> <p>Using a reverse genetics approach, we successfully isolated and identified five tRNA modification genes in <it>Arabidopsis thaliana</it>. We conclude that the method established in this study will facilitate the identification of tRNA modification genes in a wide variety of plant species.</p

Chloroplast genome sequencing analysis of Heterosigma akashiwo CCMP452 (West Atlantic) and NIES293 (West Pacific) strains

Background: Heterokont algae form a monophyletic group within the stramenopile branch of the tree of life. These organisms display wide morphological diversity, ranging from minute unicells to massive, bladed forms. Surprisingly, chloroplast genome sequences are available only for diatoms, representing two (Coscinodiscophyceae and Bacillariophyceae) of approximately 18 classes of algae that comprise this taxonomic cluster. A universal challenge to chloroplast genome sequencing studies is the retrieval of highly purified DNA in quantities sufficient for analytical processing. To circumvent this problem, we have developed a simplified method for sequencing chloroplast genomes, using fosmids selected from a total cellular DNA library. The technique has been used to sequence chloroplast DNA of two Heterosigma akashiwo strains. This raphidophyte has served as a model system for studies of stramenopile chloroplast biogenesis and evolution. Results: H. akashiwo strain CCMP452 (West Atlantic) chloroplast DNA is 160,149 bp in size with a 21,822-bp inverted repeat, whereas NIES293 (West Pacific) chloroplast DNA is 159,370 bp in size and has an inverted repeat of 21,665 bp. The fosmid cloning technique reveals that both strains contain an isomeric chloroplast DNA population resulting from an inversion of their single copy domains. Both strains contain multiple small inverted and tandem repeats, non-randomly distributed within the genomes. Although both CCMP452 and NIES293 chloroplast DNAs contains 197 genes, multiple nucleotide polymorphisms are present in both coding and intergenic regions. Several protein-coding genes contain large, in-frame inserts relative to orthologous genes in other plastids. These inserts are maintained in mRNA products. Two genes of interest in H. akashiwo, not previously reported in any chloroplast genome, include tyrC, a tyrosine recombinase, which we hypothesize may be a result of a lateral gene transfer event, and an unidentified 456 amino acid protein, which we hypothesize serves as a G-protein-coupled receptor. The H. akashiwo chloroplast genomes share little synteny with other algal chloroplast genomes sequenced to date. Conclusion: The fosmid cloning technique eliminates chloroplast isolation, does not require chloroplast DNA purification, and reduces sequencing processing time. Application of this method has provided new insights into chloroplast genome architecture, gene content and evolution within the stramenopile cluster

Importance of adenosine-to-inosine editing adjacent to the anticodon in an Arabidopsis alanine tRNA under environmental stress

In all organisms, transfer RNAs (tRNAs) undergo extensive post-transcriptional modifications. Although base modifications in the anticodon are known to alter decoding specificity or improve decoding accuracy, much less is known about the functional relevance of modifications in other positions of tRNAs. Here, we report the identification of an A-to-I tRNA editing enzyme that modifies the tRNA-Ala(AGC) in the model plant Arabidopsis thaliana. The enzyme is homologous to Tad1p, a yeast tRNA-specific adenosine deaminase, and it selectively deaminates the adenosine in the position 3'-adjacent to the anticodon (A(37)) to inosine. We show that the AtTAD1 protein is exclusively localized in the nucleus. The tad1 loss-of-function mutants isolated in Arabidopsis show normal accumulation of the tRNA-Ala(AGC), suggesting that the loss of the I(37) modification does not affect tRNA stability. The tad1 knockout mutants display no discernible phenotype under standard growth conditions, but produce less biomass under environmental stress conditions. Our results provide the first evidence in support of a physiological relevance of the A(37)-to-I modification in eukaryotes

Structural studies of the mitochondrial ribosome and co-translational insertion of membrane proteins

The mitochondrion, a constituent of eukaryotic cells is important in the synthesis of ATP through a process called oxidative phosphorylation. The enzymes that make up the subunits of oxidative phosphorylation in humans and yeast are encoded by both nuclear DNA and in mitochondrial DNA. As such the mitochondrion has retained its own translational system to include mitochondrial ribosomes (mitoribosome) to translate these proteins along with other proteins which is species dependent. The mitoribosome is also involved in co-translational insertion of proteins destined for the inner mitochondrial membrane through binding of its translocon OXA1. My study has revealed the structure of the complete 75-component yeast Saccharomyces cerevisiae mitochondrial ribosome solved to 3.3 Å by single particle cryo-electron microscopy (cryo-EM). The previously unsolved small subunit of the mitoribosome was built de novo to include 34 proteins, of which 7 are specific to the yeast mitochondrial ribosome and the 15S ribosomal RNA. This mitochondrial ribosome has a distinct architecture compared to other mitochondrial ribosome and its ancestral bacterial ribosome. Elements specific to the yeast mitoribosome give it a distinct shape and architecture to include a putatively active enzyme on the periphery. An expanded messenger RNA exit channel has also been found harbouring a platform suitable for translational activator binding. In general this structure has provided insight into translation specialisation amongst species and its continued evolution. Multiple states of actively translating human mitoribosome have been elucidated using single particle cryo-EM. The human mitoribosome structures, stalled by different antibiotics, have been seen with A/A and P/P site tRNAs in situ as well as structures with an unidentified factor in the mitoribosomal factor site. In addition the human mitochondrial large subunit and stalled complete mitoribosome has been found at low resolution likely complexed to its translocon OXA1L following detergent solubilisation.Wellcome Trust Clinical PhD Fellowship (110301/Z/15/Z