RNA editing of larch mitochondrial tRNA(His) precursors is a prerequisite for processing.
ABSTRACT: Larch mitochondria contain a'native'tRNAHis which is absent from angiosperms. Sequence comparisons of genomic DNA and cDNA obtained from unprocessed primary transcripts of the larch mitochondrial gene trnH encoding this tRNA revealed three nucleotide discrepancies. These three nucleotide alterations, in the acceptor stem, D stem and anticodon stem respectively, are conversions of genomic cytidines to thymidines in the cDNA (uridines in the tRNA) and thus resemble the RNA editing events observed in nearly all plant mitochondrial mRNAs. Two cases of editing affecting mitochondrial tRNAs from angiosperms have already been described, but we present here the first example of such events in a gymnosperm mitochondrial tRNA. All three editing events correct mismatched C x A base pairs which appear when folding the gene sequence into the standard cloverleaf structure, thereby improving the secondary structure of the tRNA. When incubated with a heterologous potato mitochondrial processing extract, only the edited form of the larch mitochondrial tRNAHis precursor was efficiently processed in vitro. These data strongly suggest that editing of larch mitochondrial tRNAHis is a prerequisite for its processing.
Project description:BACKGROUND:Plant mitochondrial genomes (mitogenomes) can be structurally complex while their size can vary from ~?222 Kbp in Brassica napus to 11.3 Mbp in Silene conica. To date, in comparison with the number of plant species, only a few plant mitogenomes have been sequenced and released, particularly for conifers (the Pinaceae family). Conifers cover an ancient group of land plants that includes about 600 species, and which are of great ecological and economical value. Among them, Siberian larch (Larix sibirica Ledeb.) represents one of the keystone species in Siberian boreal forests. Yet, despite its importance for evolutionary and population studies, the mitogenome of Siberian larch has not yet been assembled and studied. RESULTS:Two sources of DNA sequences were used to search for mitochondrial DNA (mtDNA) sequences: mtDNA enriched samples and nucleotide reads generated in the de novo whole genome sequencing project, respectively. The assembly of the Siberian larch mitogenome contained nine contigs, with the shortest and the largest contigs being 24,767?bp and 4,008,762?bp, respectively. The total size of the genome was estimated at 11.7 Mbp. In total, 40 protein-coding, 34 tRNA, and 3 rRNA genes and numerous repetitive elements (REs) were annotated in this mitogenome. In total, 864 C-to-U RNA editing sites were found for 38 out of 40 protein-coding genes. The immense size of this genome, currently the largest reported, can be partly explained by variable numbers of mobile genetic elements, and introns, but unlikely by plasmid-related sequences. We found few plasmid-like insertions representing only 0.11% of the entire Siberian larch mitogenome. CONCLUSIONS:Our study showed that the size of the Siberian larch mitogenome is much larger than in other so far studied Gymnosperms, and in the same range as for the annual flowering plant Silene conica (11.3 Mbp). Similar to other species, the Siberian larch mitogenome contains relatively few genes, and despite its huge size, the repeated and low complexity regions cover only 14.46% of the mitogenome sequence.
Project description:Human RNA methyltransferase BCDIN3D is overexpressed in breast cancer cells, and is related to the tumorigenic phenotype and poor prognosis of breast cancer. Here, we show that cytoplasmic tRNAHis is the primary target of BCDIN3D in human cells. Recombinant human BCDIN3D, expressed in Escherichia coli, monomethylates the 5΄-monophosphate of cytoplasmic tRNAHis efficiently in vitro. In BCDN3D-knockout cells, established by CRISPR/Cas9 editing, the methyl moiety at the 5΄-monophosphate of cytoplasmic tRNAHis is lost, and the exogenous expression of BCDIN3D in the knockout cells restores the modification in cytoplasmic tRNAHis. BCIDN3D recognizes the 5΄-guanosine nucleoside at position -1 (G-1) and the eight-nucleotide acceptor helix with the G-1-A73 mis-pair at the top of the acceptor stem of cytoplasmic tRNAHis, which are exceptional structural features among cytoplasmic tRNA species. While the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis affects neither the overall aminoacylation process in vitro nor the steady-state level of cytoplasmic tRNAHisin vivo, it protects the cytoplasmic tRNAHis transcript from degradation in vitro. Thus, BCDIN3D acts as a cytoplasmic tRNAHis-specific 5΄-methylphosphate capping enzyme. The present results also suggest the possible involvement of the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis and/or cytoplasmic tRNAHis itself in the tumorigenesis of breast cancer cells.
Project description:BACKGROUND:The main objectives of this study were sequencing, assembling, and annotation of chloroplast genome of one of the main Siberian boreal forest tree conifer species Siberian larch (Larix sibirica Ledeb.) and detection of polymorphic genetic markers - microsatellite loci or simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). RESULTS:We used the data of the whole genome sequencing of three Siberian larch trees from different regions - the Urals, Krasnoyarsk, and Khakassia, respectively. Sequence reads were obtained using the Illumina HiSeq2000 in the Laboratory of Forest Genomics at the Genome Research and Education Center of the Siberian Federal University. The assembling was done using the Bowtie2 mapping program and the SPAdes genomic assembler. The genome annotation was performed using the RAST service. We used the GMATo program for the SSRs search, and the Bowtie2 and UGENE programs for the SNPs detection. Length of the assembled chloroplast genome was 122,561?bp, which is similar to 122,474?bp in the closely related European larch (Larix decidua Mill.). As a result of annotation and comparison of the data with the existing data available only for three larch species - L. decidua, L. potaninii var. chinensis (complete genome 122,492?bp), and L. occidentalis (partial genome of 119,680?bp), we identified 110 genes, 34 of which represented tRNA, 4 rRNA, and 72 protein-coding genes. In total, 13 SNPs were detected; two of them were in the tRNA-Arg and Cell division protein FtsH genes, respectively. In addition, 23 SSR loci were identified. CONCLUSIONS:The complete chloroplast genome sequence was obtained for Siberian larch for the first time. The reference complete chloroplast genomes, such as one described here, would greatly help in the chloroplast resequencing and search for additional genetic markers using population samples. The results of this research will be useful for further phylogenetic and gene flow studies in conifers.
Project description:In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNAHis 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (?o) cells, we showed ?70% decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ?60% increase in aminoacylated level of tRNAHis was observed in mutant cells. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ?46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.
Project description:Mitochondrial tRNA (mt-tRNA) 5'-editing was first described more than 20 years ago; however, the first candidates for 5'-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5'-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5'-editing in D. discoideum with 5'-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5'-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5'-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species.
Project description:In contrast to biological invasions, translocations of individuals within a species range are understudied, due to difficulties in systematically detecting them. This results in limited knowledge about the corresponding processes and uncertainties regarding the status of extant populations. European larch, a forest tree whose fragmented native distribution is restricted to the Alps and to other Central European mountains, has been massively planted for at least 300 years. Here we focus on the genetic characterization of translocations having taken place within its native range. Microsatellite variation at 13 nuclear loci and sequence data of two mitochondrial DNA fragments were analyzed on the basis of a comprehensive range-wide population sample. Two complementary methods (Geneclass and Structure) were used to infer translocation events based on nuclear data whereas mitochondrial data were used for validation of these inferences. Using Geneclass, we found translocation events in a majority of populations. Additional cases of translocation and many instances of admixture were identified using Structure, thanks to the clear-cut ancestral genetic structure detected in this species. In particular, a strong divide between Alpine and Central European populations, also apparent at mitochondrial markers, helped uncover details on translocation events and related processes. Translocations and associated admixture events were found to be heterogeneously distributed across the species range, with a particularly high frequency in Central Europe. Furthermore, translocations frequently involved multiple geographic sources, some of which were over-represented. Our study illustrates the importance of range-wide investigations for tracing translocations back to their origins and for revealing some of their consequences. It provides some first clues for developing suitable conservation and management strategies.
Project description:For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3'-terminal CCA sequence that includes the site of aminoacylation, the additional 5'-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3'-CCA and 5'-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3'-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.
Project description:Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNAAla as a major determinant. However, this base pair is divergent in human mitochondrial tRNAAla, but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNAAla, in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNAAla in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg663, likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.
Project description:The larch (Larix spp.) forest in eastern Siberia is the world's largest coniferous forest. Its persistence is considered to depend on near-surface permafrost, and thus, forecast warming over the 21st century and consequent degradation of near-surface permafrost is expected to affect the larch forest in Siberia. However, predictions of these effects vary greatly, and many uncertainties remain about land - atmosphere interactions within the ecosystem. We developed an integrated land surface model to analyze how the Siberian larch forest will react to current warming trends. This model analyzed interactions between vegetation dynamics and thermo-hydrology, although it does not consider many processes those are considered to affect productivity response to a changing climate (e.g., nitrogen limitation, waterlogged soil, heat stress, and change in species composition). The model showed that, under climatic conditions predicted under gradual and rapid warming, the annual net primary production of larch increased about 2 and 3 times, respectively, by the end of the 21st century compared with that in the previous century. Soil water content during the larch-growing season showed no obvious trend, even when surface permafrost was allowed to decay and result in subsurface runoff. A sensitivity test showed that the forecast temperature and precipitation trends extended larch leafing days and reduced water shortages during the growing season, thereby increasing productivity. The integrated model also satisfactorily reconstructed latitudinal gradients in permafrost presence, soil moisture, tree leaf area index, and biomass over the entire larch-dominated area in eastern Siberia. Projected changes to ecosystem hydrology and larch productivity at this geographical scale were consistent with those from site-level simulation. This study reduces the uncertainty surrounding the impact of current climate trends on this globally important carbon reservoir, and it demonstrates the need to consider complex ecological processes to make accurate predictions.
Project description:Editing is a general event in plant mitochondrial messenger RNAs, but has never been detected in a plant mitochondrial transfer RNA (tRNA). We demonstrate here the occurrence of a tRNA editing event in higher plant mitochondria: in both bean and potato, the C encoded at position 4 in the mitochondrial tRNA(Phe)(GAA) gene is converted into a U in the mature tRNA. This nucleotide change corrects the mismatched C4-A69 base-pair which appears when folding the gene sequence into the cloverleaf structure and it is consistent with the fact that C to U transitions constitute the common editing events affecting plant mitochondrial messenger RNAs. The tRNA(Phe)(GAA) gene is located upstream of the single copy tRNA(Pro)(UGG) gene in both the potato and the bean mitochondrial DNAs. The sequences of potato and bean tRNA(Pro)(UGG) genes are colinear with the sequence of the mature bean mitochondrial tRNA(Pro)(UGG), demonstrating that this tRNA is not edited. A single copy tRNA(Ser)(GCU) gene was found upstream of the tRNA(Phe) gene in the potato mitochondrial DNA. A U6-U67 mismatched base-pair appears in the cloverleaf folding of this gene and is maintained in the mature potato mitochondrial tRNA(Ser)(GCU), which argues in favour of the hypothesis that the editing system of plant mitochondria can only perform C to U or occasionally U to C changes.