"PUS3 is a member of the highly conserved TruA/Pus3 family of pseudouridylases, which are important for healthy growth in bacteria and yeast ( Bekaert and Rousset 2005 ; Carbone et al. 1991 ; Chang et al. 1971 ; Lecointe et al. 2002 ; Tsui et al. 1991 ), and catalyze pseudouridine formation at specific uridine residues in the anticodon-stem loop of tRNAs in all kingdoms of life"

"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"

"Since tRNA Phe from LCLs with the Arg435* allele of PUS3 had almost exactly 1 mole/mole less Ψ than control LCLs, we infer that the Arg435* allele nearly completely knocks out PUS3 function due to truncation of the conserved domain and the last 45 amino acids"

"Lack of PUS3 activity is clearly physiologically consequential. Bacterial truA mutants have significant growth defects ( Chang et al. 1971 ; Tsui et al. 1991 ), while S. cerevisiae pus3Δ mutants are slow growing and temperature-sensitive ( Carbone et al. 1991 ; Lecointe et al. 2002 ), primarily due to reduced function of tRNA Gln(UUG) ( Han et al. 2015 ), and have reduced −1 frameshifting"

"it is possible that brain-specific phenotype reflects a higher sensitivity to reduced translational efficiency by the brain compared to other organs ( Torres et al. 2014 ). This would be consistent with the finding by us and others that mutations in other genes involved in tRNA modification result in phenotypes with predilection to the brain"

"The dimer interface (∼2,420 Å2) of PUS3 is exclusively formed by an anti-parallel coiled-coil domain, which is formed by a long C-terminal helix (aa 338–369) from each of the PUS3 monomers"

"PUS3WT, but not PUS3D118A, converts U39 into Ψ39 in vitro but does not modify uridines in other positions of the tRNAs (Figures 1B and S1D), demonstrating that our purified PUS3WT is functional and displays the expected target specificity"

"Each tRNA molecule in our structure contacts both PUS3 monomers and is held by the PUS3 dimer at two main contact points: the elbow region (T-arm) and the ASL"

"PUS3 fails to bind to the ASL alone, which is consistent with data for the bacterial EcTruA dimer"

"we found no evidence in human cells that PUS3 targets other RNA classes than tRNAs, including mRNAs, rRNAs, lncRNA, or small nuclear RNAs (snRNAs)"

"the structure of human PUS3 with pre-tRNAUCUArg represents the first structural snapshot of a tRNA modifying enzyme bound to an intron-containing tRNA, suggesting that PUS3 acts before the tRNA splicing endonuclease (TSEN) complex"

"we characterize two main classes of pathogenic PUS3 variants: those that reduce protein stability and others that cause defects in tRNA binding and/or modification activity"

"the purified recombinant PUS3Y71C protein shows similar binding affinities and modification activities as the wild‐type PUS3 enzyme in vitro. However, the Y71C mutation compromises the thermostability of the protein and leads to almost complete depletion of PUS3 protein levels in patient‐derived fibroblasts"

"the introduction of the Y71C mutation strongly compromises the stability of the PUS3 protein, resulting in a melting temperature that is ~10°C lower than for PUS3WT"

"the p.Tyr71Cys substitution neither affect tRNA binding nor pseudouridylation activity in vitro, but strongly impair the thermostability profile of PUS3, while the p.Ile299Thr mutation causes protein aggregation"

"Our analyses show that the levels of Ψ39 dramatically decreased on all tested tRNA transcripts from the patient samples"

"the endogenous protein level of PUS3 was significantly reduced in the patient cells... The patients and the healthy controls expressed similar levels of PUS3 mRNAs, showing that the observed drop in protein levels is caused post‐transcriptionally"

"PUS3 encodes the pseudouridylate synthase 3, an enzyme catalyzing the formation of tRNA pseudouridine, which plays a critical role in tRNA structure, function, and stability"

"Biallelic pathogenic variants of PUS3 have been previously associated with severe intellectual disability, microcephaly, epilepsy, and short stature"

"We identified a novel homozygous PUS3 frameshift variant in a child with facial dysmorphisms, growth failure, microcephaly, retinal dystrophy, cerebellar hypoplasia, congenital heart defect, and right kidney hypoplasia"

"The cryo-EM structure of human PUS3 bound to two tRNA-Gln molecules at 3.0 Å resolution shows a symmetric homodimer with each tRNA contacting both PUS3 monomers"

"The elbow region of tRNA is positioned on a platform formed by two helices containing basic residues, while the anticodon stem loop is accommodated by the second PUS3 subunit positioning U39 in the catalytic cleft"

"6% of the particles corresponded to PUS3 dimers bound to a single tRNA molecule, demonstrating asymmetric binding modes"

"The highest-confidence, PUS3-specific reaction is installation of **Ψ in the tRNA anticodon stem–loop**, particularly **positions 38/39** (often discussed as **Ψ38/Ψ39**). A primary biochemical study using recombinant human PUS3 shows direct catalysis of **tRNA Ψ39** formation in vitro using CMC-based primer extension assays."

"Mechanistic synthesis of recent work indicates that human PUS3 has **strict selectivity for intact, tRNA-shaped substrates** (recognizing global tRNA architecture) and **does not bind isolated anticodon stem-loop fragments** efficiently."

"In transcriptome-wide analyses summarized in a mechanistic review, **Pseudo-seq in PUS3-depleted human cells found no PUS3-dependent pseudouridylation sites in mRNAs**, supporting that PUS3’s primary substrates are tRNAs rather than mRNAs."

"the 2025 review describes an anti-parallel coiled-coil C-terminal helix and interaction with the tRNA elbow plus anticodon stem-loop, explaining why full tRNA architecture is needed for catalysis."

"Within the retrieved evidence set, PUS3 is reported as present in **nucleus and cytoplasm**, consistent with roles spanning nuclear pre-tRNA processing/maturation and cytoplasmic function of modified tRNAs. No retrieved evidence supported mitochondrial localization for human PUS3."

"certain disease-associated PUS3 missense variants can cause disease **not by abolishing catalytic activity in vitro**, but by **destabilizing/aggregating the enzyme**, thereby reducing cellular protein levels and lowering PUS3-dependent tRNA Ψ levels in patient cells."

"Yeast functional effects summarized qualitatively: loss of Ψ38/39 reduced stop-codon readthrough; Ψ39 was required for +1 frameshifts at slippery sequences."