Mitochondrial Genomes in Perkinsus Decode Conserved Frameshifts in All Genes.

Mitochondrial genomes of apicomplexans, dinoflagellates, and chrompodellids that collectively make up the Myzozoa, encode only three proteins (Cytochrome b [COB], Cytochrome c oxidase subunit 1 [COX1], Cytochrome c oxidase subunit 3 [COX3]), contain fragmented ribosomal RNAs, and display extensive recombination, RNA trans-splicing, and RNA-editing. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remained poorly characterized. Previous reports of Perkinsus genes indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts. To determine both ancestral myzozoan and novel perkinsozoan mitochondrial genome features, we sequenced and assembled mitochondrial genomes of four Perkinsus species. These data show a simple ancestral genome with the common reduced coding capacity but disposition for rearrangement. We identified 75 frameshifts across the four species that occur as distinct types and that are highly conserved in gene location. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. The cox3 gene is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in cox1 and cob. All genes lack cysteine codons implying a reduction to 19 amino acids in these genomes. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA suggesting import into the organelle. Perkinsus demonstrates further remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.

CCA-Addition Gone Wild: Unusual Occurrence and Phylogeny of Four Different tRNA Nucleotidyltransferases in Acanthamoeba castellanii.

tRNAs are important players in the protein synthesis machinery, where they act as adapter molecules for translating the mRNA codons into the corresponding amino acid sequence. In a series of highly conserved maturation steps, the primary transcripts are converted into mature tRNAs. In the amoebozoan Acanthamoeba castellanii, a highly unusual evolution of some of these processing steps was identified that are based on unconventional RNA polymerase activities. In this context, we investigated the synthesis of the 3'-terminal CCA-end that is added posttranscriptionally by a specialized polymerase, the tRNA nucleotidyltransferase (CCA-adding enzyme). The majority of eukaryotic organisms carry only a single gene for a CCA-adding enzyme that acts on both the cytosolic and the mitochondrial tRNA pool. In a bioinformatic analysis of the genome of this organism, we identified a surprising multitude of genes for enzymes that contain the active site signature of eukaryotic/eubacterial tRNA nucleotidyltransferases. In vitro activity analyses of these enzymes revealed that two proteins represent bona fide CCA-adding enzymes, one of them carrying an N-terminal sequence corresponding to a putative mitochondrial target signal. The other enzymes have restricted activities and represent CC- and A-adding enzymes, respectively. The A-adding enzyme is of particular interest, as its sequence is closely related to corresponding enzymes from Proteobacteria, indicating a horizontal gene transfer. Interestingly, this unusual diversity of nucleotidyltransferase genes is not restricted to Acanthamoeba castellanii but is also present in other members of the Acanthamoeba genus, indicating an ancient evolutionary trait.

LOTTE-seq (Long hairpin oligonucleotide based tRNA high-throughput sequencing): specific selection of tRNAs with 3'-CCA end for high-throughput sequencing.

Transfer RNAs belong to the most abundant type of ribonucleic acid in the cell, and detailed investigations revealed correlations between alterations in the tRNA pool composition and certain diseases like breast cancer. However, currently available methods do not sample the entire tRNA pool or lack specificity for tRNAs. A specific disadvantage of such methods is that only full-length tRNAs are analysed, while tRNA fragments or incomplete cDNAs due to RT stops at modified nucleosides are lost. Another drawback in certain approaches is that the tRNA fraction has to be isolated and separated from high molecular weight RNA, resulting in considerable labour costs and loss of material. Based on a hairpin-shaped adapter oligonucleotide selective for tRNA transcripts, we developed a highly specific protocol for efficient and comprehensive high-throughput analysis of tRNAs that combines the benefits of existing methods and eliminates their disadvantages. Due to a 3'-TGG overhang, the adapter is specifically ligated to the tRNA 3'-CCA end. Reverse transcription prior to the ligation of a second adapter allows to include prematurely terminated cDNA products, increasing the number of tRNA reads. This strategy renders this approach a powerful and universal tool to analyse the tRNA pool of cells and organisms under different conditions in health and disease.

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes.

Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

Unusual Occurrence of Two Bona-Fide CCA-Adding Enzymes in Dictyostelium discoideum.

Dictyostelium discoideum, the model organism for the evolutionary supergroup of Amoebozoa, is a social amoeba that, upon starvation, undergoes transition from a unicellular to a multicellular organism. In its genome, we identified two genes encoding for tRNA nucleotidyltransferases. Such pairs of tRNA nucleotidyltransferases usually represent collaborating partial activities catalyzing CC- and A-addition to the tRNA 3'-end, respectively. In D. discoideum, however, both enzymes exhibit identical activities, representing bona-fide CCA-adding enzymes. Detailed characterization of the corresponding activities revealed that both enzymes seem to be essential and are regulated inversely during different developmental stages of D. discoideum. Intriguingly, this is the first description of two functionally equivalent CCA-adding enzymes using the same set of tRNAs and showing a similar distribution within the cell. This situation seems to be a common feature in Dictyostelia, as other members of this phylum carry similar pairs of tRNA nucleotidyltransferase genes in their genome.

Cold adaptation of tRNA nucleotidyltransferases: A tradeoff in activity, stability and fidelity.

Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.

Expression of the Arabidopsis redox-related LEA protein, SAG21 is regulated by ERF, NAC and WRKY transcription factors.

SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.

Changes of the tRNA Modification Pattern during the Development of Dictyostelium discoideum.

Dictyostelium discoideum is a social amoeba, which on starvation develops from a single-cell state to a multicellular fruiting body. This developmental process is accompanied by massive changes in gene expression, which also affect non-coding RNAs. Here, we investigate how tRNAs as key regulators of the translation process are affected by this transition. To this end, we used LOTTE-seq to sequence the tRNA pool of D. discoideum at different developmental time points and analyzed both tRNA composition and tRNA modification patterns. We developed a workflow for the specific detection of modifications from reverse transcriptase signatures in chemically untreated RNA-seq data at single-nucleotide resolution. It avoids the comparison of treated and untreated RNA-seq data using reverse transcription arrest patterns at nucleotides in the neighborhood of a putative modification site as internal control. We find that nucleotide modification sites in D. discoideum tRNAs largely conform to the modification patterns observed throughout the eukaroytes. However, there are also previously undescribed modification sites. We observe substantial dynamic changes of both expression levels and modification patterns of certain tRNA types during fruiting body development. Beyond the specific application to D. discoideum our results demonstrate that the developmental variability of tRNA expression and modification can be traced efficiently with LOTTE-seq.