Origin, evolution, and mechanism of 5' tRNA editing in chytridiomycete fungi

5' tRNA editing has been demonstrated to occur in the mitochondria of the distantly related rhizopod amoeba Acanthamoeba castellanii and the chytridiomycete fungus Spizellomyces punctatus. In these organisms, canonical tRNA structures are restored by removing mismatched nucleotides at the first three 5' positions and replacing them with nucleotides capable of forming Watson-Crick base pairs with their 3' counterparts. This form of editing seems likely to occur in members of Amoebozoa other than A. castellanii, as well as in members of Heterolobosea. Evidence for 5' tRNA editing has not been found to date, however, in any other fungus including the deeply branching chytridiomycete Allomyces macrogynus. We predicted that a similar form of tRNA editing would occur in members of the chytridiomycete order Monoblepharidales based on the analysis of complete mitochondrial tRNA complements. This prediction was confirmed by analysis of tRNA sequences using a tRNA circularization/RT-PCR-based approach. The presence of partially and completely unedited tRNAs in members of the Monoblepharidales suggests the involvement of a 5'-to-3' exonuclease rather than an endonuclease in removing the three 5' nucleotides from a tRNA substrate. Surprisingly, analysis of the mtDNA of the chytridiomycete Rhizophydium brooksianum, which branches as a sister group to S. punctatus in molecular phylogenies, did not suggest the presence of editing. This prediction was also confirmed experimentally. The absence of tRNA editing in R. brooksianum raises the possibility that 5' tRNA editing may have evolved twice independently within Chytridiomycota, once in the lineage leading to S. punctatus and once in the lineage leading to the Monoblepharidales.     

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.     

End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae

All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNA(Trp)) is edited by a C to U modification at the first position of the anticodon. To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNA(Trp)(CCA), tRNA(Ile)(UAU), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile) and tRNA(Lys) were mainly mitochondrial; and tRNA(Trp) and tRNA(Val) were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNA(Ile) underwent importation in vitro into isolated mitochondria more efficiently than 5'-extended precursor tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNA(Trp) was imported in vitro more efficiently than edited tRNA(Trp), suggesting the presence of importation determinants in the anticodon.     

tRNA 3' processing in plants: nuclear and mitochondrial activities differ

The nuclear tRNA 3' processing activity from wheat has been characterized and partially purified. Several characteristics of the wheat nuclear 3' processing enzyme now allow this activity to be distinguished from its mitochondrial counterpart. The nuclear enzyme is an endonuclease, which we termed nuclear RNase Z. The enzyme cleaves at the discriminator base and seems to consist only of protein subunits, since essential RNA subunits could not be detected. RNase Z leaves 5' terminal phosphoryl and 3' terminal hydroxyl groups at the processing products. It is a stable enzyme being active over broad temperature and pH ranges, with the highest activity at 35 degrees C and pH 8.4. The apparent molecular mass according to gel filtration chromatography is 122 kDa. The nuclear RNase Z does process 5' extended pretRNAs but with a much lower efficiency than 5' matured pretRNAs. Nuclear intron-containing precursor tRNAs as well as mitochondrial precursor tRNAs are efficiently cleaved by the nuclear RNase Z. Mitochondrial pretRNA(His) is processed by the nuclear RNase Z, generating a mature tRNA(His) containing an 8 base pair acceptor stem. The edited mitochondrial pretRNA(Phe) is cleaved easily, while the unedited version having a mismatch in the acceptor stem is not cleaved. Thus, an intact acceptor stem seems to be required for processing. Experiments with precursors containing mutated tRNAs showed that a completely intact anticodon arm is not necessary for processing by RNase Z. Comparison of the plant nuclear tRNA 3' processing enzyme with the plant mitochondrial one suggests that both activities are different enzymes.     

A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5'-tRNA editing

Genes with sequence similarity to the yeast tRNA(His) guanylyltransferase (Thg1) gene have been identified in all three domains of life, and Thg1 family enzymes are implicated in diverse processes, ranging from tRNA(His) maturation to 5'-end repair of tRNAs. All of these activities take advantage of the ability of Thg1 family enzymes to catalyze 3'-5' nucleotide addition reactions. Although many Thg1-containing organisms have a single Thg1-related gene, certain eukaryotic microbes possess multiple genes with sequence similarity to Thg1. Here we investigate the activities of four Thg1-like proteins (TLPs) encoded by the genome of the slime mold, Dictyostelium discoideum (a member of the eukaryotic supergroup Amoebozoa). We show that one of the four TLPs is a bona fide Thg1 ortholog, a cytoplasmic G(-1) addition enzyme likely to be responsible for tRNA(His) maturation in D. discoideum. Two other D. discoideum TLPs exhibit biochemical activities consistent with a role for these enzymes in mitochondrial 5'-tRNA editing, based on their ability to efficiently repair the 5' ends of mitochondrial tRNA editing substrates. Although 5'-tRNA editing was discovered nearly two decades ago, the identity of the protein(s) that catalyze this activity has remained elusive. This article provides the first identification of any purified protein that appears to play a role in the 5'-tRNA editing reaction. Moreover, the presence of multiple Thg1 family members in D. discoideum suggests that gene duplication and divergence during evolution has resulted in paralogous proteins that use 3'-5' nucleotide addition reactions for diverse biological functions in the same organism.     

Escherichia coli 16S rRNA 3''-end formation requires a distal transfer RNA sequence at a proper distance.

The 16S rRNA species in bacterial precursor rRNAs is followed by two evolutionarily conserved features: (i) a double-stranded stem formed by complementary sequences adjacent to the 5' and 3' ends of the 16S rRNA; and (ii) a 3'-transfer RNA sequence. To assess the possible role of these features, plasmid constructs with precursor-specific features deleted were tested for their capacity to form mature rRNA. Stem-forming sequences were dispensable for both 5' and 3' terminus formation; whereas an intact spacer tRNA positioned greater than 24 nucleotides downstream of the 16S RNA sequence was required for correct 3'-end maturation. These results suggest that spacer tRNA at an appropriate location helps form a conformation obligate for pre-rRNA processing, perhaps by binding to a nascent binding site in preribosomes. Thus, spacer tRNAs may be an obligate participant in ribosome formation.     

Hyaloraphidium curvatum: a linear mitochondrial genome, tRNA editing, and an evolutionary link to lower fungi

We have sequenced the mitochondrial DNA (mtDNA) of Hyaloraphidium curvatum, an organism previously classified as a colorless green alga but now recognized as a lower fungus based on molecular data. The 29.97-kbp mitochondrial chromosome is maintained as a monomeric, linear molecule with identical, inverted repeats (1.43 kbp) at both ends, a rare genome architecture in mitochondria. The genome encodes only 14 known mitochondrial proteins, 7 tRNAs, the large subunit rRNA and small subunit rRNA (SSU rRNA), and 3 ORFs. The SSU rRNA is encoded in two gene pieces that are located 8 kbp apart on the mtDNA. Scrambled and fragmented mitochondrial rRNAs are well known from green algae and alveolate protists but are unprecedented in fungi. Protein genes code for apocytochrome b; cytochrome oxidase 1, 2, and 3, NADH dehydrogenase 1, 2, 3, 4, 4L, 5, and 6, and ATP synthase 6, 8, and 9 subunits, and several of these genes are organized in operon-like clusters. The set of seven mitochondrially encoded tRNAs is insufficient to recognize all codons that occur in the mitochondrial protein genes. When taking into account the pronounced codon bias, at least 16 nuclear-encoded tRNAs are assumed to be imported into the mitochondria. Three of the seven predicted mitochondria-encoded tRNA sequences carry mispairings in the first three positions of the acceptor stem. This strongly suggests that these tRNAs are edited by a mechanism similar to the one seen in the fungus Spizellomyces punctatus and the rhizopod amoeba Acanthamoeba castellanii. Our phylogenetic analysis confirms with overwhelming support that H. curvatum is a member of the chytridiomycete fungi, specifically related to the Monoblepharidales.     

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.     

Length and Sequence Heterogeneity in the Mitochondrial Internal Transcribed Spacer of Acanthamoeba spp.

ABSTRACT. The internal transcribed spacer (ITS) between the mitochondrial large (23S rRNA; rnl ) and small (16S rRNA; rns ) subunit ribosomal RNA genes of Acanthamoeba castellanii strain Neff was sequenced previously and was uniquely interesting because it contained tRNA genes with acceptor stem mismatches that underwent RNA editing repair. Our interest in this ITS region was to determine its phylogenetic potential in differentiating between closely related isolates. We analyzed the mitochondrial ITS region for 17 Acanthamoeba isolates and observed extensive sequence and length variability, making this region difficult to align. Acanthamoeba griffini strain S-7 had the shortest ITS (i.e. 559 base pairs [bp]) compared with Acanthamoeba palestinensis strain Reich, which had the longest (i.e. 1,360 bp). The length disparity occurred predominantly between the spacer region of the aspartic acid ( trnD ) and methionine ( trnM ) tRNA genes. Unexpectedly, this region in A. palestinensis Reich was found to contain a duplication of the trnM gene. Additionally, like A. castellanii strain Neff, all isolates examined had tRNAs with mismatches in their acceptor stem. Also, the potential for an additional type of editing not described previously for Acanthamoeba , involving purine to pyrimidine transversions was observed.     

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa.

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).     

The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria.

Recent advances in in vitrosystems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3'-end of gRNA to the editing site appear to be untenable. Two types of in vitrosystems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3'-end of the 5'-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3'-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitroevidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.