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

Abstract

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.

FIGURE 1.

Sequence alignment of D. discoideum TLPs with Thg1 family members. Sequences of the four D. discoideum TLPs (DdiTLP1–4) were aligned with representative Thg1/TLP sequences from Eucarya (human; HsThg1 and Saccharomyces cerevisiae; ScThg1), Archaea (M. acetivorans; MaTLP and M. thermoautotrophicus; MtTLP) and Bacteria (Bacillus. thuringiensis; BtTLP) using Multalin (Corpet 1988). Dark and light shading indicate >90% and >50% sequence identity, respectively. Numbers in parentheses at the end of the sequence are the number of additional C-terminal residues not shown in the alignment. Residues highlighted in red are putative N-terminal mitochondrial targeting sequences predicted by MitoProt (Claros 1995; Claros and Vincens 1996). The Q/N-rich domain found in DdiTLP3 is indicated by underlining; M107 is indicated by a black star. Red stars indicate universally conserved residues that are critical for Thg1/TLP function (Jackman and Phizicky 2008). Residues indicated with blue stars are catalytically important for the activity of yeast Thg1, but are conserved only in eukaryotic Thg1 family members (including DdiTLP1). This includes the highly conserved HINNLYN motif (underlined in blue) that is catalytically critical for yeast Thg1 activity but is not found in bacterial or archaeal Thg1 enzymes sequences.

FIGURE 2.

DdiTLP1 exhibits Thg1 activity in vitro. (A) SDS-PAGE analysis of purified TLPs from D. discoideum. Purified proteins (3 μg each) were resolved by 15% SDS-PAGE; lane MW, molecular weight markers. Predicted molecular weights: TLP1, 32 kDa; TLP2 (without mitochondrial peptide), 32 kDa; TLP3, 50 kDa, and TLP4, 36 kDa. (B) G₋₁ addition activity tested with 5′-³²P-labeled yeast tRNA^His using each of the purified D. discoideum TLPs and yeast Thg1. Lane +, G₋₁ addition product generated by yeast Thg1; −, no enzyme control. The phosphatase protection assay was performed on reactions containing decreasing amounts of each purified protein (fivefold serial dilutions, see Materials and Methods for exact concentrations of each protein). The trimeric G₋₁ addition product (Gp*GpC) was resolved from inorganic phosphate (P*_i) derived from remaining substrate using silica TLC. Specific activities for DdiTLP1 and yeast Thg1 were calculated by quantification of the amount of purified protein that catalyzed 10% conversion of substrate to G₋₁-containing product in 2 h at room temperature (defined as 1 Unit of enzyme activity); specific activities (SA) were expressed as Units per microgram of purified protein and indicated below each panel.

FIGURE 3.

DdiTLP1 catalyzes Thg1 activity in vivo in yeast. Complementation of the yeast thg1Δ growth defect by each of the D. discoideum TLPs was tested using a plasmid shuffle assay, as previously described (Abad et al. 2010). DdiTLPs, as indicated, expressed under control of a galactose-inducible promoter on LEU2 plasmids, were transformed into the yeast thg1Δ strain and two independent positive transformants were tested by replica plating onto the indicated media. Clones of TLP2 and TLP3 lacked their respective mitochondrial targeting sequences and the Q/N-rich sequence for TLP3 (see Fig. 1). Empty vector control strains (vect) were also tested. Photographs were taken after 3–4 d growth at 30°C.

FIGURE 4.

Mitochondrial tRNA sequences from D. discoideum indicate a need for 5′-tRNA editing. Aminoacyl-acceptor stems of the 18 mt-tRNAs encoded in the D. discoideum mitochondrial genome are shown; the remainder of each tRNA sequence is omitted for clarity. Two genes for mt-tRNA^Ile_GAU that differ by only two nucleotides are indicated as Ile_GAU1/2 as they are identical in their aminoacyl-acceptor stems. The need for mt-tRNA editing is reflected by the presence of non-Watson–Crick unpaired residues in the acceptor stem for the nine tRNAs (including the two Ile_GAU isoforms) shown on the bottom row. Residues expected to be added by 5′ editing are indicated by arrows to the left of each incorrect nucleotide.

FIGURE 5.

DdiTLP3 and DdiTLP4 repair 5′-truncated mt-tRNA^Ile_CAU. (A) Schematic of phosphatase protection assay used with 5′-³²P-labeled tRNA to detect G₊₁ addition catalyzed by TLPs in the presence of ATP (0.1 mM) and GTP (1 mM). Only aminoacyl-acceptor stem sequences of the tRNAs are shown for clarity; lines indicate connection to the tRNA body. Expected products of RNase A/CIP treatment are shown beneath each tRNA. (B) The addition of G₊₁ assayed as shown in A with decreasing concentrations of each purified TLP (fivefold serial dilutions of each). Identities of the labeled species are indicated to the left of the figure. Lane −, no-enzyme control reaction. The maximum percent reaction products (sum of G₊₁ and AppG reaction products, if observed) formed by TLP2, -3, and -4 is indicated below each panel.

FIGURE 6.

DdiTLP3 and DdiTLP4 add a missing C₊₂ to 5′-truncated mt-tRNA^Leu_UAG. (A) Schematic of phosphatase protection assay used with 5′-³²P-labeled tRNA to detect addition catalyzed by TLPs in the presence of ATP (0.1 mM) and CTP (1 mM). Only aminoacyl-acceptor stem sequences are shown for clarity and reaction products of RNase T1/CIP treatment are shown below each tRNA sequence. (B) 3′-5′ Addition activity was tested using the assay shown in A with fivefold serial dilutions of each of the four purified TLPs. Lane Y, yeast Thg1 (1 μg), lane −, no-enzyme control. The major reaction products produced by TLP3 and TLP4 are indicated as P1 and P2; the identities of these products were assigned as shown in parentheses based on further nuclease digestion. (C) Analysis of P1 and P2 reaction products of DdiTLP4 reaction with 5′-truncated mt-tRNA^Leu_UAG. The TLC-purified P1 and P2 products (shown in the first lane of each panel, labeled −) were tested by further nuclease digestion using RNAse T1 (T1), RNase T2 (T2), nuclease P1 (P1), and RNase A (A), as indicated. The expected reaction products for each digestion are shown below each panel. Products were resolved by PEI-cellulose TLC; positions of cold NMP standards (C, A, U, and G) were visualized by UV shadowing and are indicated by circles to the left of the figure.

FIGURE 7.

DdiTLP4 (and DdiTLP3) fully repair 5′-truncated mt-tRNA^Leu_UAG using 3′-5′ polymerization. (A) Schematic of nuclease digestion assay used with 5′-³²P-labeled tRNA to detect addition catalyzed by TLPs in the presence of various nucleotides, as indicated. The relative thicknesses of the reaction arrows reflect the experimentally observed (see B, C below) preference for the addition of templated G₊₁ over the addition of nontemplated A₊₁, observed when GTP was omitted from the assays. For this assay, reactions were treated with phosphatase and purified by phenol extraction and ethanol precipitation prior to further nuclease digestion; expected reaction products for each tRNA are shown by dashed arrows. (B) Assays contained DdiTLP4 (30 μM), or no enzyme (−) as indicated, and either CTP only (1 mM), CTP (1 mM) + ATP (0.1 mM), or all three NTPs (1 mM each CTP and GTP, and 0.1 mM ATP), and were digested with the indicated nuclease. Reaction products were resolved using PEI-cellulose TLC; positions of unlabeled NMP standards (C, A, U, and G) were visualized by UV shadowing and are indicated to the left of the figure. Identical results were observed in reactions with DdiTLP3, not shown here for clarity. (C) Reaction products from panel B were resolved by silica TLC to further separate products that remained at the origin following RNase A digestion. Identities of labeled species are indicated to the right of the figure.