Autosomal-recessive cerebellar atrophy is usually associated with inactivating mutations and early-onset presentation. The underlying molecular diagnosis suggests the involvement of neuronal survival pathways, but many mechanisms are still lacking and most patients elude genetic diagnosis. Using whole exome sequencing, we identified homozygous p.Val55Ala in the THG1L (tRNA-histidine guanylyltransferase 1 like) gene in three siblings who presented with cerebellar signs, developmental delay, dysarthria, and pyramidal signs and had cerebellar atrophy on brain MRI. THG1L protein was previously reported to participate in mitochondrial fusion via its interaction with MFN2. Abnormal mitochondrial fragmentation, including mitochondria accumulation around the nuclei and confinement of the mitochondrial network to the nuclear vicinity, was observed when patient fibroblasts were cultured in galactose containing medium. Culturing cells in galactose containing media promotes cellular respiration by oxidative phosphorylation and the action of the electron transport chain thus stimulating mitochondrial activity. The growth defect of the yeast thg1Δ strain was rescued by the expression of either yeast Thg1 or human THG1L; however, clear growth defect was observed following the expression of the human p.Val55Ala THG1L or the corresponding yeast mutant. A defect in the protein tRNAHis guanylyltransferase activity was excluded by the normal in vitro G−1 addition to either yeast tRNAHis or human mitochondrial tRNAHis in the presence of the THG1L mutation. We propose that homozygosity for the p.Val55Ala mutation in THG1L is the cause of the abnormal mitochondrial network in the patient fibroblasts, likely by interfering with THG1L activity towards MFN2. This may result in lack of mitochondria in the cerebellar Purkinje dendrites, with degeneration of Purkinje cell bodies and apoptosis of granule cells, as reported for MFN2 deficient mice.

In eukaryotes, the tRNAHis guanylyltransferase (Thg1) catalyzes 3′–5′ addition of a single guanosine residue to the −1 position (G–1) of tRNAHis, across from a highly conserved adenosine at position 73 (A73). After addition of G–1, Thg1 removes pyrophosphate from the tRNA 5′-end, generating 5′-monophosphorylated G–1-containing tRNA. The presence of the 5′-monophosphorylated G–1 residue is important for recognition of tRNAHis by its cognate histidyl-tRNA synthetase. In addition to the single-G–1 addition reaction, Thg1 polymerizes multiple G residues to the 5′-end of tRNAHis variants. For 3′–5′ polymerization, Thg1 uses the 3′-end of the tRNAHis acceptor stem as a template. The mechanism of reverse polymerization is presumed to involve nucleophilic attack of the 3′-OH from each incoming NTP on the intact 5′-triphosphate created by the preceding nucleotide addition. The potential exists for competition between 5′-pyrophosphate removal and 3′–5′ polymerase reactions that could define the outcome of Thg1-catalyzed addition, yet the interplay between these competing reactions has not been investigated for any Thg1 enzyme. Here we establish transient kinetic assays to characterize the pyrophosphate removal versus nucleotide addition activities of yeast Thg1 with a set of tRNAHis substrates in which the identity of the N–1:N73 base pair was varied to mimic various products of the N–1 addition reaction catalyzed by Thg1. We demonstrate that retention of the 5′-triphosphate is correlated with efficient 3′–5′ reverse polymerization. A kinetic partitioning mechanism that acts to prevent addition of nucleotides beyond the −1 position with wild-type tRNAHis is proposed.

The tRNAHis guanylyltransferase (Thg1) catalyzes the incorporation of a single guanosine residue at the −1 position (G–1) of tRNAHis, using an unusual 3′–5′ nucleotidyl transfer reaction. Thg1 and Thg1 orthologs known as Thg1-like proteins (TLPs), which catalyze tRNA repair and editing, are the only known enzymes that add nucleotides in the 3′–5′ direction. Thg1 enzymes share no identifiable sequence similarity with any other known enzyme family that could be used to suggest the mechanism for catalysis of the unusual 3′–5′ addition reaction. The high-resolution crystal structure of human Thg1 revealed remarkable structural similarity between canonical DNA/RNA polymerases and eukaryotic Thg1; nevertheless, questions regarding the molecular mechanism of 3′–5′ nucleotide addition remain. Here, we use transient kinetics to measure the pseudo-first-order forward rate constants for the three steps of the G–1 addition reaction catalyzed by yeast Thg1: adenylylation of the 5′ end of the tRNA (kaden), nucleotidyl transfer (kntrans), and removal of pyrophosphate from the G–1-containing tRNA (kppase). This kinetic framework, in conjunction with the crystal structure of nucleotide-bound Thg1, suggests a likely role for two-metal ion chemistry in all three chemical steps of the G–1 addition reaction. Furthermore, we have identified additional residues (K44 and N161) involved in adenylylation and three positively charged residues (R27, K96, and R133) that participate primarily in the nucleotidyl transfer step of the reaction. These data provide a foundation for understanding the mechanism of 3′–5′ nucleotide addition in tRNAHis maturation.

The presence of an additional 5′ guanosine residue (G-1) is a unique feature of tRNAHis. G-1 is incorporated posttranscriptionally in eukarya via an unusual 3′–5′ nucleotide addition reaction catalyzed by the tRNAHis guanylyltransferase (Thg1). Yeast Thg1 catalyzes an unexpected second activity: Watson–Crick-dependent 3′–5′ nucleotide addition that occurs in the opposite direction to nucleotide addition by all known DNA and RNA polymerases. This discovery led to the hypothesis that there are alternative roles for Thg1 family members that take advantage of this unusual enzymatic activity. Here we show that archaeal homologs of Thg1 catalyze G-1 addition, in vitro and in vivo in yeast, but only in a templated reaction, i.e. with tRNAHis substrates that contain a C73 discriminator nucleotide. Because tRNAHis from archaea contains C73, these findings are consistent with a physiological function for templated nucleotide addition in archaeal tRNAHis maturation. Moreover, unlike yeast Thg1, archaeal Thg1 enzymes also exhibit a preference for template-dependent U-1 addition to A73-containing tRNAHis. Taken together, these results demonstrate that Watson–Crick template-dependent 3′–5′ nucleotide addition is a shared catalytic activity exhibited by Thg1 family members from multiple domains of life, and therefore, that this unusual reaction may constitute an ancestral activity present in the earliest members of the Thg1 enzyme family.

All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5′ to 3′ direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNAHis guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3′-5′ addition of nucleotides to nucleic acid substrates. The 2.3-Å crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5′-3′ DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5′-3′ and 3′-5′ reactions raises important questions concerning selection of the 5′-3′ mechanism during the evolution of nucleotide polymerases.

The yeast tRNAHis guanylyltransferase (Thg1) is an essential enzyme in yeast. Thg1 adds a single G residue to the 5′ end of tRNAHis (G−1), which serves as a crucial determinant for aminoacylation of tRNAHis. Thg1 is the only known gene product that catalyzes the 3′−5′ addition of a single nucleotide via a normal phosphodiester bond, and since there is no identifiable sequence similarity between Thg1 and any other known enzyme family, the mechanism by which Thg1 catalyzes this unique reaction remains unclear. We have altered 29 highly conserved Thg1 residues to alanine, and using three assays to assess Thg1 catalytic activity and substrate specificity, we have demonstrated that the vast majority of these highly conserved residues (24/29) affect Thg1 function in some measurable way. We have identified 12 Thg1 residues that are critical for G−1 addition, based on significantly decreased ability to add G−1 to tRNAHis in vitro and significant defects in complementation of a thg1Δ yeast strain. We have also identified a single Thg1 alteration (D68A) that causes a dramatic decrease in the rigorous specificity of Thg1 for tRNAHis. This single alteration enhances the kcat/KM for ppp-tRNAPhe by nearly 100-fold relative to that of wild-type Thg1. These results suggest that Thg1 substrate recognition is at least in part mediated by preventing recognition of incorrect substrates for nucleotide addition.

•Mitochondrial tRNA in many protozoan eukaryotes undergo 5′-editing.•TLPs use 3′–5′ polymerase activity to repair 5′-truncated editing substrates.•A. castellanii TLP2 tolerates G–U pairs in editing substrates better than other TLPs.•TLP activities correlate with biological outcomes for G–U base pairs during editing.•Biochemical evidence suggests specialized functions for different eukaryotic TLPs.Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5′-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G–U base pairs, wherein some species remove G–U base pairs during 5′-editing, while others retain G–U pairs in the edited tRNA. We tested whether 3′–5′ polymerases that catalyze the repair step of 5′-editing exhibit organism-specific preferences that explain the treatment of G–U base pairs. Biochemical and kinetic approaches revealed that a 3′–5′ polymerase from Acanthamoeba castellanii tolerates G–U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.

•Mitochondrial tRNA in many protozoan eukaryotes undergo 5′-editing.•TLPs use 3′–5′ polymerase activity to repair 5′-truncated editing substrates.•A. castellanii TLP2 tolerates G–U pairs in editing substrates better than other TLPs.•TLP activities correlate with biological outcomes for G–U base pairs during editing.•Biochemical evidence suggests specialized functions for different eukaryotic TLPs.Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5′-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G–U base pairs, wherein some species remove G–U base pairs during 5′-editing, while others retain G–U pairs in the edited tRNA. We tested whether 3′–5′ polymerases that catalyze the repair step of 5′-editing exhibit organism-specific preferences that explain the treatment of G–U base pairs. Biochemical and kinetic approaches revealed that a 3′–5′ polymerase from Acanthamoeba castellanii tolerates G–U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.