The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Destabilization of mutated human PUS3 protein causes intellectual disability.

Pseudouridine (Ψ) is an RNA base modification ubiquitously found in many types of RNAs. In humans, the isomerization of uridine is catalyzed by different stand-alone pseudouridine synthases (PUS). Genomic mutations in the human pseudouridine synthase 3 gene (PUS3) have been identified in patients with neurodevelopmental disorders. However, the underlying molecular mechanisms that cause the disease phenotypes remain elusive. Here, we utilize exome sequencing to identify genomic variants that lead to a homozygous amino acid substitution (p.[(Tyr71Cys)];[(Tyr71Cys)]) in human PUS3 of two affected individuals and a compound heterozygous substitution (p.[(Tyr71Cys)];[(Ile299Thr)]) in a third patient. We obtain wild-type and mutated full-length human recombinant PUS3 proteins and characterize the enzymatic activity in vitro. Unexpectedly, we find that 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. Concomitantly, we observe that the PUS3 protein levels as well as the level of PUS3-dependent Ψ levels are strongly reduced in fibroblasts derived from all three patients. In summary, our results directly illustrate the link between the identified PUS3 variants and reduced Ψ levels in the patient cells, providing a molecular explanation for the observed clinical phenotypes.

PUS3-related disorder: Report of a novel patient and delineation of the phenotypic spectrum.

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. This patient further expands the phenotypic spectrum of PUS3-related disorders to include a more severe syndromic presentation.

Clinical and molecular delineation of PUS3-associated neurodevelopmental disorders.

Biallelic variants in PUS3 have recently been recognized as a rare cause of neurodevelopmental disorders. Pseudouridine synthase-3 encoded by PUS3 is an enzyme important for modification of various RNAs, including transfer RNA (tRNA). Here we present the clinical and genetic features of 21 individuals with biallelic PUS3 variants: seven new and 14 previously reported individuals, where clinical features of two were updated. The clinical and genetic information were collected through collaborations or by literature search. All individuals were characterized by the local clinicians and the gene variants were identified by next generation sequencing (NGS) based methodologies. The clinical picture was dominated by global developmental delay, epilepsy, hypotonia and microcephaly. Gray sclera, which has previously been suggested to be a characteristic feature of PUS3-associated phenotypes, was reported in only seven individuals. The patients had some dysmorphic facial features, but a recognizable gestalt was not observed. In conclusion, homozygous and compound heterozygous PUS3 variants lead to a rare neurodevelopmental disorder. Further functional studies are necessary to understand involvement of PUS3 and tRNA biogenesis in normal and abnormal brain development.

Differentiating the Roles of UL16, UL21, and Us3 in the Nuclear Egress of Herpes Simplex Virus Capsids.

Viral proteins pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex virus 2 capsids. To better understand the role of these proteins in nuclear egress, we established whether nuclear egress complex (NEC) distribution and/or function was altered in the absence of either pUL16 or pUL21. NEC distribution in cells infected with pUL16-deficient viruses was indistinguishable from that observed in cells infected with wild-type viruses. In contrast, NEC distribution was aberrant in cells infected with pUL21-deficient virus and, instead, showed some similarity to the aberrant NEC distribution pattern observed in cells infected with pUs3-deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher-resolution examination of nuclear envelope ultrastructure in cells infected with pUL21-deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21-deficient virus infections was dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16, or both pUs3 and pUL16 were consistent with a role for pUL16 in advance of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress.IMPORTANCE The membrane deformation activity of the herpesvirus nuclear egress complex (NEC) allows capsids to transit through both nuclear membranes into the cytoplasm. NEC activity must be precisely controlled during viral infection, and yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus 2, function in nuclear egress, we examined how the lack of each protein impacted NEC distribution. These analyses revealed a function of pUL16 in nuclear egress distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity, and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of capsids is required for all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process and aid the development of herpes-specific antiviral therapies.

Genetic basis of neurodevelopmental disorders in 103 Jordanian families.

We recruited 103 families from Jordan with neurodevelopmental disorders (NDD) and patterns of inheritance mostly suggestive of autosomal recessive inheritance. In each family, we investigated at least one affected individual using exome sequencing and an in-house diagnostic variant interpretation pipeline including a search for copy number variation. This approach led us to identify the likely molecular defect in established disease genes in 37 families. We could identify 25 pathogenic nonsense and 11 missense variants as well as 3 pathogenic copy number variants and 1 repeat expansion. Notably, 11 of the disease-causal variants occurred de novo. In addition, we prioritized a homozygous frameshift variant in PUS3 in two sisters with intellectual disability. To our knowledge, PUS3 has been postulated only recently as a candidate disease gene for intellectual disability in a single family with three affected siblings. Our findings provide additional evidence to establish loss of PUS3 function as a cause of intellectual disability.

Roles of the Different Isoforms of the Pseudorabies Virus Protein Kinase pUS3 in Nuclear Egress.

Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from betaherpesviruses and gammaherpesviruses. pUS3 homologs are multifunctional and are involved in many processes, including modification of the cytoskeleton, inhibition of apoptosis, and immune evasion. pUS3 also plays a role in efficient nuclear egress of alphaherpesvirus nucleocapsids. In the absence of pUS3, primary enveloped virions accumulate in the perinuclear space (PNS) in large invaginations of the inner nuclear membrane (INM), pointing to a modulatory function for pUS3 during deenvelopment. The HSV and pseudorabies virus (PrV) US3 genes are transcribed into two mRNAs encoding two pUS3 isoforms, which have different aminoterminal sequences and abundances. To test whether the two isoforms in PrV serve different functions, we constructed mutant viruses expressing exclusively either the larger minor or the smaller major isoform, a mutant virus with decreased expression of the smaller isoform, or a mutant with impaired kinase function. Respective virus mutants were investigated in several cell lines. Our results show that absence of the larger pUS3 isoform has no detectable effect on viral replication in cell culture, while full expression of the smaller isoform and intact kinase activity is required for efficient nuclear egress. Absence of pUS3 resulted in only minor titer reduction in most cell lines tested but disclosed a more severe defect in Madin-Darby bovine kidney cells. However, accumulations of primary virions in the PNS do not account for the observed titer reduction in PrV.IMPORTANCE A plethora of substrates and functions have been assigned to the alphaherpesviral pUS3 kinase, including a role in nuclear egress. In PrV, two different pUS3 isoforms are expressed, which differ in size, abundance, and intracellular localization. Their respective role in replication is unknown, however. Here, we show that efficient nuclear egress of PrV requires the smaller isoform and intact kinase activity, whereas absence of the larger isoform has no significant effect on viral replication. Thus, there is a clear distinction in function between the two US3 gene products of PrV.

Role of Pseudouridine Formation by Deg1 for Functionality of Two Glutamine Isoacceptor tRNAs.

Loss of Deg1/Pus3 and concomitant elimination of pseudouridine in tRNA at positions 38 and 39 (ψ38/39) was shown to specifically impair the function of tRNAGlnUUG under conditions of temperature-induced down-regulation of wobble uridine thiolation in budding yeast and is linked to intellectual disability in humans. To further characterize the differential importance of the frequent ψ38/39 modification for tRNAs in yeast, we analyzed the in vivo function of non-sense suppressor tRNAs SUP4 and sup70-65 in the absence of the modifier. In the tRNATyrGψA variant SUP4, UAA read-through is enabled due to an anticodon mutation (UψA), whereas sup70-65 is a mutant form of tRNAGlnCUG (SUP70) that mediates UAG decoding due to a mutation of the anticodon-loop closing base pair (G31:C39 to A31:C39). While SUP4 function is unaltered in deg1/pus3 mutants, the ability of sup70-65 to mediate non-sense suppression and to complement a genomic deletion of the essential SUP70 gene is severely compromised. These results and the differential suppression of growth defects in deg1 mutants by multi-copy SUP70 or tQ(UUG) are consistent with the interpretation that ψ38 is most important for tRNAGlnUUG function under heat stress but becomes crucial for tRNAGlnCUG as well when the anticodon loop is destabilized by the sup70-65 mutation. Thus, ψ38/39 may protect the anticodon loop configuration from disturbances by loss of other modifications or base changes.

Functional importance of Ψ38 and Ψ39 in distinct tRNAs, amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast.

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U38 and U39 in the anticodon stem-loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ38 or Ψ39 is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA(Gln(UUG)), which normally has Ψ38. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s(2)U moiety of mcm(5)s(2)U34 of tRNA(Gln(UUG)) and the other two cytoplasmic species with mcm(5)s(2)U34, that the reduced s(2)U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s(2)U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ38,39 provides evidence that Ψ38 is important for function of tRNA(Gln(UUG)) at permissive temperature, and indicates that Ψ39 is important for the function of tRNA(Trp(CCA)) in trm10Δ pus3Δ mutants and of tRNA(Leu(CAA)) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ38 and Ψ39 in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.