A role for tRNAHis guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5′-tRNA editing – PMC

The enzyme Thg1 (tRNAHis guanylyltransferase), whose gene was originally ... DdiTLP1 was still ∼10-fold less active than yeast Thg1 in this assay, 
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The enzyme Thg1 (tRNAHis guanylyltransferase), whose gene was originally identified in yeast (Gu et al. 2003), is part of a family of enzymes found in all three domains of life and united by the ability to catalyze the 3′-5′ addition of nucleotides to nucleic acid substrates. Thg1 is widely distributed throughout eukaryotes (domain Eucarya), where it uses 3′-5′ nucleotide addition to attach a single essential guanylate residue (G−1) to the 5′ end of tRNAHis species (Cooley et al. 1982; Gu et al. 2005; Preston and Phizicky 2010). The presence of G−1 is unique to tRNAHis and acts as a necessary recognition element for efficient histidylation, both in vitro and in vivo in yeast (Rudinger et al. 1994, 1997; Nameki et al. 1995; Rosen and Musier-Forsyth 2004). The biological functions of Thg1-like proteins (TLPs) found in the domains Bacteria and Archaea are less well understood. In some archaeons, such as Methanobacterium thermoautotrophicus and Methanopyrus kandleri, TLPs likely add the required G−1 to tRNAHis, similar to their eukaryotic counterparts (Abad et al. 2010; Heinemann et al. 2010). However, in other archaeons, such as Methanosarcina acetivorans and Methanosarcina barkeri, and in nearly all bacterial species, tRNAHis genes already contain an encoded G−1 residue. The genomically encoded G−1 is presumably incorporated into the precursor tRNA during transcription, and can be retained in the mature tRNAHis due to an unusual 5′-end processing reaction catalyzed by RNase P, as occurs in Escherichia coli (Orellana et al. 1986). The lack of an obligate requirement for post-transcriptional G−1 addition in these Thg1-containing species suggests that some Thg1 family members may use 3′-5′ addition for alternative functions, beyond a simple role in tRNAHis maturation.

Thg1/TLP family members from Archaea and Bacteria exhibit at least two biochemical differences from eukaryotic family members studied so far (Abad et al. 2010; Rao et al. 2010). First, unlike yeast Thg1, bacterial/archaeal TLPs only add Watson-Crick base-paired nucleotides in the 3′-5′ addition reaction (Abad et al. 2010). Template-dependent 3′-5′ addition activity appears to be an ancestral property, as Thg1/TLP enzymes from all domains of life, including eukaryotes, catalyze this reaction (Jackman and Phizicky 2006b; Heinemann et al. 2009, 2010; Abad et al. 2010; Rao et al. 2010). Second, TLPs from Bacteria and Archaea exhibit a strong kinetic preference for the templated addition of nucleotides to the 5′ end of truncated tRNA substrates over the addition of −1 nucleotides to full-length tRNAs, and they add the missing 5′-nucleotides to tRNAs other than tRNAHis (Rao et al. 2010). Neither the addition to 5′-truncated tRNAs nor the addition to other tRNAs is catalyzed efficiently by yeast Thg1. The ability of bacterial/archaeal TLPs to restore a fully base-paired aminoacyl stem suggests a role for TLPs in previously unidentified pathways of tRNA 5′-end repair. Interestingly, TLPs from Archaea that require post-transcriptional G−1 addition to tRNAHis also catalyze the 5′-end repair reaction, suggesting that some Thg1 family members may participate in both tRNAHis maturation and general tRNA repair activities (Rao et al. 2010).

5′-End repair of tRNA is also an essential component of a 5′-tRNA editing activity that occurs in the mitochondria of a number of distantly related eukaryotic microbes (Lonergan and Gray 1993a, b; Laforest et al. 1997, 2004; Price and Gray 1999a; Gott et al. 2010). In these protists, such as the amoebozoon Acanthamoeba castellanii, where 5′-tRNA editing was first discovered, certain mitochondrially encoded tRNAs are predicted to contain up to three mismatched nucleotides at the first three positions in their aminoacyl-acceptor stem. The 5′-tRNA editing activity is thought to comprise at least two components: first, a nucleolytic activity that removes the incorrectly paired nucleotides from the 5′ end of the tRNA, and second, a 3′-5′ nucleotide addition activity that restores the missing nucleotides, using the 3′ end of the tRNA as the template to generate a fully paired aminoacyl-acceptor stem (Price and Gray 1999b; Bullerwell and Gray 2005). Although 5′-tRNA editing was the first of a number of tRNA editing activities to be described, the identity of the protein(s) that catalyze this reaction has remained unknown. However, the striking similarity of the 3′-5′ addition component of the 5′-tRNA editing reaction to the 5′-end repair activity recently associated with bacterial and archaeal TLPs suggested that enzymes from the Thg1 superfamily could play a role in 5′-tRNA editing.

In support of this hypothesis, multiple Thg1-related sequences have been identified in the genomes of several protists predicted to require 5′-tRNA editing to produce functional mitochondrial tRNAs. BLAST searches using yeast Thg1 have revealed the presence of two Thg1-related sequences in A. castellanii and in the chytid fungus, Spizellomyces punctatus (unpublished results), as well as four different genes with sequence similarity to yeast Thg1 in another amoebozoon, the slime mold Dictyostelium discoideum (Altschul et al. 1997; Fey et al. 2009). We chose the D. discoideum enzymes for further investigation based on the availability of a well-characterized genomic assembly for this organism and because of its biochemical tractability. Interestingly, one of the four D. discoideum sequences (initially called DdiTLP1 and here renamed DdiThg1) shares more sequence similarity with eukaryal Thg1 enzymes, such as yeast Thg1 (54% identity/72% similarity between DdiTLP1 and yeast Thg1), than with prokaryal TLPs, such as M. acetivorans TLP (30% identity/47% similarity between DdiTLP1 and archaeal MaTLP), consistent with a function for DdiTLP1 in cytoplasmic tRNAHis maturation (Fig. 1). The other three DdiTLPs (DdiTLP2-4) share more sequence identity with bacterial and archaeal family members (Fig. 1), suggesting that one or more of these could be components of the 5′-tRNA editing enzyme. Two of the DdiTLPs (TLP2 and TLP3) are predicted to contain mitochondrial targeting sequences, which would also be consistent with a role for these enzymes in mitochondrial 5′-tRNA editing.

To test these hypotheses, we characterized the biochemical activities of each of the four Thg1-related sequences found in D. discoideum. Here we demonstrate that the bona fide Thg1 ortholog from Ddi (DdiThg1) adds G−1 to tRNAHis, both in vitro and in vivo consistent with its predicted function from sequence analysis and predicted cytoplasmic localization. Using an assay for 3′-5′ nucleotide addition to 5′-truncated mitochondrial-tRNA (mt-tRNA) substrates from D. discoideum, we also show that two of the TLPs (DdiTLP3 and DdiTLP4) exhibit biochemical activities consistent with their participation in editing. These results constitute the first identification of any purified protein that could play a role in 5′-tRNA editing in eukaryotes. As 5′-tRNA editing reactions require the addition of any of four nucleotides to complete the acceptor stem of substrate tRNAs, and occur with many different mt-tRNA species, this observation suggests more generalized roles for 3′-5′ nucleotide addition than the simple addition of a single nucleotide to tRNAHis first associated with members of this enzyme family in yeast and multicellular eukaryotes.

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