Nuclear-encoded tRNA Research Articles

Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss.

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.

Functional characterization of the human tRNA methyltransferases TRMT10A and TRMT10B.

The TRM10 family of methyltransferases is responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukarya. The human genome encodes three TRM10-type enzymes, of which only the mitochondrial TRMT10C was previously characterized in detail, whereas the functional significance of the two presumably nuclear enzymes TRMT10A and TRMT10B remained unexplained. Here we show that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear-encoded tRNA. Furthermore, we show that the lack of G9 methylation causes a decrease in the steady-state levels of the initiator tRNAiMet-CAT and an alteration in its further post-transcriptional modification. Our work finally clarifies the function of TRMT10A and TRMT10B in vivo and provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs required for protein synthesis.

Molecular characterization of three PRORP proteins in the moss Physcomitrella patens: nuclear PRORP protein is not essential for moss viability.

RNase P is a ubiquitous endonuclease that removes the 5′ leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5′-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5′-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNAArg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNAArg (CCG) and pre-tRNAArg (UCU) but they cleaved pre-tRNAArg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.

Identification of highly-disrupted tRNA genes in nuclear genome of the red alga, Cyanidioschyzon merolae 10D

The limited locations of tRNA introns are crucial for eukaryal tRNA-splicing endonuclease recognition. However, our analysis of the nuclear genome of an early-diverged red alga, Cyanidioschyzon merolae, demonstrated the first evidence of nuclear-encoded tRNA genes that contain ectopic and/or multiple introns. Some genes exhibited both intronic and permuted structures in which the 3′-half of the tRNA coding sequence lies upstream of the 5′-half, and an intron is inserted into either half. These highly disrupted tRNA genes, which account for 63% of all nuclear tRNA genes, are expressed via the orderly and sequential processing of bulge-helix-bulge (BHB) motifs at intron-exon junctions and termini of permuted tRNA precursors, probably by a C. merolae tRNA-splicing endonuclease with an unidentified subunit architecture. The results revealed a considerable diversity in eukaryal tRNA intron properties and endonuclease architectures, which will help to elucidate the acquisition mechanism of the BHB-mediated disrupted tRNA genes.

An Incompatibility between a Mitochondrial tRNA and Its Nuclear-Encoded tRNA Synthetase Compromises Development and Fitness in Drosophila

Mitochondrial transcription, translation, and respiration require interactions between genes encoded in two distinct genomes, generating the potential for mutations in nuclear and mitochondrial genomes to interact epistatically and cause incompatibilities that decrease fitness. Mitochondrial-nuclear epistasis for fitness has been documented within and between populations and species of diverse taxa, but rarely has the genetic or mechanistic basis of these mitochondrial–nuclear interactions been elucidated, limiting our understanding of which genes harbor variants causing mitochondrial–nuclear disruption and of the pathways and processes that are impacted by mitochondrial–nuclear coevolution. Here we identify an amino acid polymorphism in the Drosophila melanogaster nuclear-encoded mitochondrial tyrosyl–tRNA synthetase that interacts epistatically with a polymorphism in the D. simulans mitochondrial-encoded tRNATyr to significantly delay development, compromise bristle formation, and decrease fecundity. The incompatible genotype specifically decreases the activities of oxidative phosphorylation complexes I, III, and IV that contain mitochondrial-encoded subunits. Combined with the identity of the interacting alleles, this pattern indicates that mitochondrial protein translation is affected by this interaction. Our findings suggest that interactions between mitochondrial tRNAs and their nuclear-encoded tRNA synthetases may be targets of compensatory molecular evolution. Human mitochondrial diseases are often genetically complex and variable in penetrance, and the mitochondrial–nuclear interaction we document provides a plausible mechanism to explain this complexity.

The Human Mitochondrial Transcriptome

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).

The ins and outs of tRNA transport

Transfer RNAs (tRNAs) are involved in many cellular functions distributed throughout the cellular space. In addition to their role in protein translation, these molecules are engaged in tasks as diverse as the regulation of gene expression, amino‐acid synthesis, protein degradation, cell‐wall synthesis, porphyrin biosynthesis, priming of replication, RNA interference and the transport of macromolecules. The nucleus and the mitochondria of eukaryotic cells, as well as the chloroplasts in plants, contain their own genome that encodes a range of proteins and nucleic acids. Early evidence suggested that some of the RNA content of mitochondria could be transcribed from non‐mitochondrial DNA (Suyama, 1967). It is now well established that a variable number of tRNA species present in the mitochondria are indeed nucleus‐encoded (Fig 1). The phenomenon of mitochondrial tRNA import has been reported in plants, marsupials, the yeast Saccharomyces cerevisiae , and the protozoa Leishmania , Trypanosoma and Tetrahymena . When certain tRNA species are not encoded by the mitochondrial genome, there is a predictable requirement for the import of nucleus‐encoded tRNAs into the mitochondria (Schneider & Marechal‐Drouard, 2000). This is the case, for example, for the mitochondria of Leishmania , which are completely devoid of tRNA‐encoding genes. However, there are a few examples of tRNA import into mitochondria that already have a complete set of mitochondria‐encoded tRNAs. Other tRNA transport systems have also been identified (Fig 1), which include their retrograde transport from the cytoplasm to the nucleus (Takano et al , 2005; Shaheen & Hopper, 2005), the export of mitochondria‐encoded tRNA to the cytoplasm (Maniataki & Mourelatos, 2005), and the packaging of tRNAs into retroviruses (Waters & Mullin, 1977). The roles of some of these transport systems have yet to be defined. Figure 1. The travels of transfer RNA. …

Accumulation of nuclear-encoded tRNA Thr (AGU) in mitochondria of the liverwort Marchantia polymorpha

The mitochondria) genome of the liverwort Marchantia polymorpha does not encode the full complement of tRNAs for the threonine and isoleucine codon boxes. To find the missing tRNA genes specifically for tRNA Thr in mitochondria, we have searched the genomic library and identified two clones (pTT1 and pTT2), encoding the identical tRNA Thr (AGU) gene copy with different 5′- and 3′-flanking sequences. By northern analysis, we demonstrate considerable accumulation of the nuclear encoded tRNA Thr and moderate expression of native tRN Thr(GGU) in mitochondria. Nonetheless, the imported and native tRNA Thr species together are not sufficient to translate all four threonine codons used in liverwort mitochondria, implicating mitochondria) import of at least one additional threonine isoacceptor tRNA.

Occurrence of nuclear-encoded tRNAIle in mitochondria of the liverwort Marchantia polymorpha.

Although 29 tRNA genes have been deduced from the complete nucleotide sequence of the mitochondrial genome from the liverwort Marchantia polymorpha, a tRNAIle gene decoding AUU and AUC codons is conspicuously absent. In order to address the question of the possible involvement of nuclear-encoded tRNA, we isolated and identified three variant copies of the nuclear-encoded tRNAIle(AAU) gene from the liverwort. Northern analysis showed the presence of nuclear-encoded tRNAIle both in the mitochondrion and the cytosol, while both chloroplast DNA-encoded tRNAIle and nuclear-encoded tRNATyr were absent in liverwort mitochondria. These results unequivocally establish that import of nuclear tRNAIle into mitochondria indeed occurs in one of the most primitive plants, M. polymorpha.

Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

Cytoplasmic tRNA(Lys)CUU is the only nuclear-encoded tRNA of Saccharomyces cerevisiae found to be associated with mitochondria. Selective import of this tRNA into isolated organelles requires cytoplasmic factors. Here we identify two of these factors as the cytoplasmic and mitochondrial lysyl-tRNA synthetases. The cytoplasmic enzyme is obligatory for in vitro import of the deacylated, but not of the aminoacylated tRNA. We thus infer that it is needed for aminoacylation of the tRNA, which is a prerequisite for its import. The mitochondrial synthetase, which cannot aminoacylate tRN(Lys)CUU, is required for import of both aminoacylated and deacylated forms. Its depletion leads to a total arrest of tRNA import, in vitro and in vivo. The mitochondrial lysyl-tRNA synthetase is able to form specific and stable RNP complexes with the amino-acylated tRNA. Furthermore, an N-terminal truncated form of the synthetase which cannot be targeted into mitochondria is unable to direct the import of the tRNA. We therefore hypothesize that the cytosolic precursor form of the mitochondrial synthetase has a carrier function for translocation of the tRNA across the mitochondrial membranes. However, cooperation of the two synthetases is not sufficient to direct tRNA import, suggesting the need of additional factor(s).