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Franz et al. J Transl Genet Genom 2020;4:50-70 I https://doi.org/10.20517/jtgg.2020.13 Page 59

PUS1, PUS3 and PUS7
Pseudouridine is a common tRNA modification, and to date, three ID proteins that play a role in
pseudouridinylation have been identified. These are PUS1, PUS3 and PUS7 (pseudouridine synthases),
which are involved in the conversion of uridine to pseudouridine at different specific tRNA positions.

Yeast Pus1 was the first eukaryotic tRNA pseudouridine synthase to be characterized and shown to be
involved in the conversion of tRNA uridines at multiple positions of introns containing tRNA Ile[196] . Pus1
targets both cytoplasmic and mitochondrial tRNA [112] and was later shown also to target U2 snRNA in
yeast [115] .

The first reported human PUS1 mutation was a homozygous missense change (R116W) found in all affected
individuals in two Italian families who suffered from mitochondrial myopathy and sideroblastic anemia
(MLASA; MIM 600462) but without ID [113] . tRNA pseudouridinylation was later shown to be greatly reduced
in patient cell lines [116] . PUS1-dependent ID was first reported in a patient with the same (R116W) missense
change by Zeharia et al. [117] . In two brothers with MLASA and a truncating PUS1 mutation (E220X), one had
ID whereas the other had an elevated intelligence quotient above normal levels [114] .

PUS3 is a pseudouridine synthase, originally isolated from yeast, that catalyzes pseudouridine formation at
positions 38 and 39 in the anticodon stem of certain tRNAs. Yeast Pus3 deletion strains are viable but grow
slowly, especially at elevated temperatures [197] . The protein was found to be evolutionarily conserved, and like
mouse Pus3, it can convert uridine at position 38 or 39 to pseudouridine in yeast and human tRNA in vitro,
albeit with different efficiency [109] .

ID caused by PUS3 deficiency is inherited as an autosomal recessive disorder. The first report of PUS3
mutations described 3 affected sisters that were homozygous for the nonsense mutation c.1303C>T, R435X,
and the phenotype in these patients was largely brain specific [110] . A second report presented a single child
from consanguineous parents, carrying a frameshift mutation (c.1181_1182delCT, Ser394CysfsTer18) and
no detectable PUS3 transcript. The child suffered from ID, microcephaly, hypotonia, seizures, and vision
[56]
and hearing loss . Furthermore, two compound heterozygous mutations were reported in a Brazilian and
a Chinese family [198,199] . Although all reported patients presented with additional features, ID was the only
consistent characteristic.

PUS7 is a multi-substrate pseudouridine synthase that in yeast targets several tRNA uridines at position 13,
Tyr
the pre-tRNA at position 35 [200] , small nucleolar RNA U2 (U2 snRNA) at position 35 [201] and also 5S and
5.8S rRNA [202] and mRNA [203] . Interestingly, uridine conversion of snRNA U2 at positions 56 and 93 can be
induced in yeast by nutrient deprivation or heat shock [204] . In human stem cells, PUS7 pseudouridinylation
was found to activate small tRNA-derived fragments that inhibit protein synthesis by targeting the initiation
complex. PUS7 inactivation leads to defective germ layer specification [205] .

Homozygous truncating PUS7 mutations were recently reported to cause ID with speech delay, short stature,
microcephaly, and aggressive behavior in patients from three different families [118] . Two ID families with
homozygous PUS7 mutations, a missense change or a deletion leading to a frameshift, were also reported.
The patients also suffered from microcephaly, whereas short stature was not seen in all patients [111] . Recently,
another ID family of Afghan origin was reported, carrying a Gly128Arg missense change. The phenotype
of the patient was milder without microcephaly or short stature, but still with speech delay and aggressive
behavior [119] . In this last study, pseudouridine levels were not investigated [119] , whereas markedly reduced
pseudouridine levels at tRNA position 13 were found in all investigated ID patients [111,118] .

Variable arm
The variable arm of tRNAs is located between the anticodon (or C) and the T arms. The length of the
variable arm depends on the tRNA and can be between 3 and 21 nucleotides long. Generally speaking, class