Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG 'stop' codons recognized as leucine.

The mitochondrial DNA of the chytridiomycete fungus Spizellomyces punctatusen codes only eight tRNAs, although a minimal set of 24-25 tRNAs is normally found in fungi. One of these tRNAs has a CAU anticodon and is structurally related to leucine tRNAs, which would permit the translation of the UAG 'stop' codons that occur in most of its protein genes. The predicted structures of all S. punctatus tRNAs have the common feature of containing one to three mis-pairings in the first three positions of their acceptor stems. Such mis-pairing is expected to impair proper folding and processing of tRNAs from their precursors. Five of these eight RNAs were shown to be edited at the RNA level, in the 5'portion of the molecules. These changes include both pyrimidine to purine and A to G substitutions that restore normal pairing in the acceptor stem. Editing was not found at other positions of the tRNAs, or in the mitochondrial mRNAs of S. punctatus. While tRNA editing has not been observed in other fungi, the editing pattern inS.punctatus is virtually identical to that described in the amoeboid protozoan Acanthamoeba castellanii. If this type of mitochondrial tRNA editing has originated from their common ancestor, one has to assume that it was independently lost in plants, animals and in most fungi. Alternatively, editing might have evolved independently, or the genes coding for the components of the editing machinery were laterally transferred.     

In vitro RNA editing in plant mitochondria does not require added energy

RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.     

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre- and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2' or 3' hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.     

In vitro RNA editing in pea mitochondria requires NTP or dNTP,suggesting involvement of an RNA helicase

To analyze the biochemical parameters of RNA editing in plant mitochondria and to eventually characterize the enzymes involved we developed a novel in vitro system. The high sensitivity of the mismatch-specific thymine glycosylase is exploited to facilitate reliable quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly processes a C to U editing site in the cognate atp9 template. Reaction conditions were determined for a number of parameters, which allow first conclusions on the proteins involved. The apparent tolerance against specific Zn2+ chelators argues against the involvement of a cytidine deaminase enzyme, the theoretically most straightforward catalysator of the deamination reaction. Participation of a transaminase was investigated by testing potential amino group receptors, but none of these increased the RNA editing reaction. Most notable is the requirement of the RNA editing activity for NTPs. Any NTP or dNTP can substitute for ATP to the optimal concentration of 15 mm. This observation suggests the participation of an RNA helicase in the predicted RNA editing protein complex of plant mitochondria.     

In vitro conversion of a methionine to a glutamine-acceptor tRNA

A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.     

In vitro antifungal activity of antifungalmycin 702, a new polyene macrolide antibiotic, against the rice blast fungus Magnaporthe grisea

Antifungalmycin 702, a novel polyene macrolide antibiotic produced by Streptomyces padanus JAU4234, strongly inhibited mycelial growth of the rice blast fungus, Magnaporthe grisea, with EC₅₀ of 37 μg/ml and EC₉₀ of 136 μg/ml. Significant reduction in the number of conidia was observed at above 20 μg/ml. Conidia germination and appressorium formation were also suppressed and were not viable with >40 μg/ml. When treated with antifungalmycin 702, hyphae morphology became irregular. Based on microscopic examination, antifungalmycin 702 may exert its antifungal activity by changing the structure of cell membranes and the cytoskeleton and interacting with the organelles. Antifungalmycin 702 thus has potential as a new fungicide in the treatment of rice blast disease.     

In vitro activity characterization of the tomato SnRK1 complex proteins

Plant Sucrose non-Fermenting 1-Related Protein Kinase1 (SnRK1) complexes are members of the Snf1/AMPK/SnRK protein kinase family and play important roles in many aspects of metabolism. In tomato (Solanum lycopersicum, Sl), only one α-subunit of the SnRK1 complex, SlSnRK1.1, has been characterized to date. In this study, the phylogenetic placement and in vitro kinase activity of a second tomato SnRK1 α-subunit, SlSnRK1.2, were characterized. Interestingly, in the phylogenetic analysis of SnRK1 sequences from monocots and dicots SlSnRK1.2 clusters only with other Solanaceae SnRK1.2 sequences, suggesting possible functional divergence of these kinases from other SnRK1 kinases. For analysis of kinase activity, SlSnRK1.2 was able to autophosphorylate, phosphorylate the complex β-subunits, and phosphorylate the SnRK1 AMARA peptide substrate, all with drastically lower overall kinase activity compared to SlSnRK1.1. Activation by the upstream kinase SlSnAK was able to increase the kinase activity of both SlSnRK1.1 and SlSnRK1.2, although the increase is less dramatic for SlSnRK1.2. The highest kinase activity on the AMARA peptide for SlSnRK1.2 was seen when reconstituting the complex in vitro with SlSip1 as the β-subunit. In comparison, SlSnRK1.1 showed the lowest kinase activity on the AMARA peptide when SlSip1 was used. These studies suggest the SlSnRK1.2 phylogenetic divergence and lower SlSnRK1.2 kinase activity compared to SlSnRK1.1 may be indicative of different in vivo roles for each kinase.     

Errors from selective disruption of the editing center in a tRNA synthetase

Some aminoacyl-tRNA synthetases have two catalytic centers that together achieve fine-structure discrimination of closely similar amino acids. The role of tRNA is to stimulate translocation of a misactivated amino acid from the active site to the editing site where the misactivated substrate is eliminated by hydrolysis. Using isoleucyl-tRNA synthetase as an example, we placed mutations in the catalytic center for editing at residues strongly conserved from bacteria to humans. A particular single substitution and one double substitution resulted in production of mischarged tRNA, by interfering specifically with the chemical step of hydrolytic editing. The substitutions affected neither amino acid activation nor aminoacylation, with the cognate amino acid. Thus, because of the demonstrated functional independence of the two catalytic sites, errors of aminoacylation can be generated by selective mutations in the center for editing.     

Insertional editing in mitochondria of Physarum

RNA produced from a number of genes on the mitochondrial (mt) DNA of Physarum polycephalum have nucleotides inserted at specific sites in their sequence. These insertions are spaced at approximately 25 nucleotide intervals and create open reading frames in mRNA and functional structure in tRNAs and rRNAs. Although most of the insertions at a site are single cytidines; single uridines and certain dinucleotides containing adenosine and guanosine as well as cytidine and uridine are also occasionally inserted at certain sites. This mixed nucleotide insertional RNA editing is unique among currently characterized editing systems.     

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).     

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria: In search of the editosome

The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a ∼1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (L*a) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (L*b) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The L*b-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and L*b-complex gradient fractions were similar in composition and L*b-complex bands often degraded to L-complex bands after manipulation or freeze–thaw cycles. The L*a-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The L*b-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the L*b-complexes. The MRP1/2 RNA binding complex is localized mainly in the L*b-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the L*b-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or “editosome.”     

In vitro characterization of a koala retrovirus

Recently, a new endogenous koala gammaretrovirus, designated KoRV, was isolated from koalas. The KoRV genome shares 78% nucleotide identity with another gammaretrovirus, gibbon ape leukemia virus (GALV). KoRV is endogenous in koalas, while GALV is exogenous, suggesting that KoRV predates GALV and that gibbons and koalas acquired the virus at different times from a common source. We have determined that subtle adaptive differences between the KoRV and GALV envelope genes account for differences in their receptor utilization properties. KoRV represents a unique example of a gammaretrovirus whose envelope has evolved to allow for its expanded host range and zoonotic potential.     

An in vitro evolved precursor tRNA with aminoacylation activity

A set of catalysts for aminoacyl-tRNA synthesis is an essential component for translation. The RNA world hypothesis postulates that RNA catalysts could have played this role. Here we show an in vitro evolved precursor tRNA consisting of two domains, a catalytic 5'-leader sequence and an aminoacyl-acceptor tRNA. The 5'-leader sequence domain selectively self-charges phenylalanine on the 3'-terminus of the tRNA domain. This cis-acting ribozyme is susceptible to RNase P RNA, generating the corresponding 5'-leader segment and the mature tRNA. Moreover, the 5'-leader segment is able to aminoacylate the mature tRNA in trans. Mutational studies have revealed that C(74) and C(75) at the tRNA aminoacyl-acceptor end form base pairs with G71 and G70 of the trans-acting ribozyme. Such Watson-Crick base pairing with tRNA has been observed in RNase P RNA and 23S rRNA, suggesting that all three ribozymes use a similar mechanism for the recognition of the aminoacyl-acceptor end. Our demonstrations indicate that catalytic precursor tRNAs could have provided the foundations for the genetic coding system in the proto-translation system.     

In vitro processing of E. coli tRNA precursors

Using E. coli tRNA precursors isolated from an RNAase P mutant strain, we have studied the steps required for the formation of tRNAs having a mature primary sequence in vitro. Our results suggest that at least three different enzymatic activities can participate in the processing of tRNA precursors.     

Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria

Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the ‘antisense’ tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs.