Intellectual disability is a common and highly heterogeneous disorder etiologically. In a multiplex consanguineous family, we applied autozygosity mapping and exome sequencing and identified a novel homozygous truncating mutation in PUS3 that fully segregates with the intellectual disability phenotype. Consistent with the known role of Pus3 in isomerizing uracil to pseudouridine at positions 38 and 39 in tRNA, we found a significant reduction in this post-transcriptional modification of tRNA in patient cells. Our finding adds to a growing list of intellectual disability disorders that are caused by perturbation of various tRNA modifications, which highlights the sensitivity of the brain to these highly conserved processes.

Yeast tRNA^His guanylyltransferase, Thg1, is an essential protein that adds a single guanine to the 5′ end (G₋₁) of tRNA^His. This G₋₁ residue is required for aminoacylation of tRNA^His by histidyl-tRNA synthetase, both in vitro and in vivo. The guanine nucleotide addition reaction catalyzed by Thg1 extends the polynucleotide chain in the reverse (3′-5′) direction of other known polymerases, albeit by one nucleotide. Here, we show that alteration of the 3′ end of the Thg1 substrate tRNA^His unleashes an unexpected reverse polymerase activity of wild-type Thg1, resulting in the 3′-5′ addition of multiple nucleotides to the tRNA, with efficiency comparable to the G₋₁ addition reaction. The addition of G₋₁ forms a mismatched G·A base pair at the 5′ end of tRNA^His, and, with monophosphorylated tRNA substrates, it is absolutely specific for tRNA^His. By contrast, reverse polymerization forms multiple G·C or C·G base pairs, and, with preactivated tRNA species, it can initiate at positions other than −1 and is not specific for tRNA^His. Thus, wild-type Thg1 catalyzes a templated polymerization reaction acting in the reverse direction of that of canonical DNA and RNA polymerases. Surprisingly, Thg1 can also readily use dNTPs for nucleotide addition. These results suggest that 3′-5′ polymerization represents either an uncharacterized role for Thg1 in RNA or DNA repair or metabolism, or it may be a remnant of an earlier catalytic strategy used in nature.

Despite the universality of tRNA modifications, some tRNAs lacking specific modifications are subject to degradation pathways, while other tRNAs lacking the same modifications are resistant. Here, we suggest a model in which some modifications have minor, possibly redundant, roles in specific tRNAs. This model is consistent with the low specificity of some modification enzymes. Limitations of this model include the limited assays and growth conditions on which these conclusions are based, as well as the high specificity exhibited by many modification enzymes with important roles in translation. The specificity of these enzymes is often enhanced by complex substrate recognition patterns and sub-cellular compartmentalization.