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

Abstract

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.

FIGURE 1.

Phylogenetic distribution of 5′ tRNA editing. (A) Schematic tree based on the branching order of published phylogenetic trees (Forget et al. 2002; Bullerwell et al. 2003a). The branching order of the animal, fungal, and amoebozoan lineages is consistent with phylogenies based on nucleus-encoded protein sequence data (Baldauf et al. 2000). Red asterisks mark the presumed, independent origins of 5′ tRNA editing. (B) Editing status of tRNAs in chytridiomycete mtDNAs. ^atRNAs where editing is predicted or confirmed/total. (Open box) tRNA gene not present; ((Black filled box) tRNA gene present, editing not predicted; (red filled box) tRNA gene present, editing confirmed; (red filled circle) tRNA gene present, editing predicted but not confirmed by sequencing; (blue filled circle) tRNA gene present, confirmed not editing.

FIGURE 2.

Partially and completely unedited monoblepharidalean mitochondrial tRNAs. (A) cDNA sequencing gels of unedited and partially edited tRNAs from Monoblepharella15. Sequence of the first three 5′ nucleotides of tRNA acceptor stems is indicated to the left of the sequencing gels, and the inferred structure of the circularized tRNA is indicated below each gel. Positions where a nucleotide change has occurred relative to the genomic sequence are indicated by arrows. (B) tRNA acceptor stems for all examples where partial or incomplete editing is observed. Predicted nucleotide substitutions are indicated by arrows, and the percentage of cDNA clones in which the predicted change occurs is indicated in brackets.

FIGURE 3.

Model of tRNA processing and editing in monoblepharidalean mitochondria. A hypothetical unprocessed tRNA acceptor stem is shown. (A) 5′ and 3′ extensions are first removed by nuclease activities (the order of events is not known). (B) CCA tails are added by nucleotidyl transferase (CCAse) and a 5′-to-3′ exonuclease activity removes nucleotides from tRNA 5′ ends. The CCAse and exonuclease activities apparently function independently of one another. A presumed 3′-to-5′ nucleotidyltransferase (as proposed for A. castellanii based on in vitro results; Price and Gray 1999b) then fills in nucleotides removed by the 5′-to-3′ exonuclease. (C) If all three 5′ nucleotides have been removed by the exonuclease, three nucleotides are added that can form WC base pairs, resulting in a completely edited tRNA. (D) If only one or two 5′ nucleotides have been removed, a partially edited tRNA results. (E) If no nucleotides have been removed, an unedited tRNA results.