Destabilization of mutated human PUS3 protein causes intellectual disability

Abstract

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

Lipidome remodeling in response to nutrient replenishment requires the tRNA modifier Deg1/Pus3 in yeast

“…The tRNA modifier Deg1 is conserved from yeast to humans and mutation of the human DEG1 orthologue PUS3 induces neurodegenerative defects partly resembling those described in patients with defects in the tRNA modification complex Elongator (Abdelrahman et al, 2018;Gaik, Kojic, Stegeman, et al, 2022;Gaik, Kojic, Wainwright, et al, 2022;Lin et al, 2022;Shaheen et al, 2016).…”

“…The tRNA modifier Deg1 is conserved from yeast to humans and mutation of the human DEG1 orthologue PUS3 induces neurodegenerative defects partly resembling those described in patients with defects in the tRNA modification complex Elongator (Abdelrahman et al., 2018; Gaik, Kojic, Stegeman, et al., 2022; Gaik, Kojic, Wainwright, et al., 2022; Lin et al., 2022; Shaheen et al., 2016). In yeast, pleiotropic phenotypes of Elongator and deg1 mutants also overlap, including the induction of protein aggregates and high LD content (Bozaquel‐Morais et al., 2018; Khonsari et al., 2021; Nedialkova & Leidel, 2015).…”

“…Deg1 is a tRNA pseudouridine synthase specifically modifying tRNA positions 38 and 39 (Lecointe et al, 1998). Absence of functional Deg1 causes pleiotropic phenotypes including slow growth and temperature sensitivity in yeast, and mutation of the human orthologue Pus3 is linked to neurodevelopmental disorders (Abdelrahman et al, 2018;Han et al, 2015;Lecointe et al, 1998;Lin et al, 2022;Shaheen et al, 2015Shaheen et al, , 2016. For yeast, pseudouridylation of tRNA-Gln UUG by Deg1 is critical to maintain full decoding efficiency, but does not affect the cellular levels of this tRNA (Han et al, 2015;Klassen et al, 2016).…”

Pseudouridine and N1-methylpseudouridine as potent nucleotide analogues for RNA therapy and vaccine development

“…16 Aberrant pseudouridine modifications in tRNA can impact translation accuracy and efficiency, potentially contributing to disease pathogenesis. 10,11,[17][18][19][20] For example, mutations in the PUS1 gene, which encodes a pseudouridine synthase, are associated with mitochondrial myopathy and sideroblastic anaemia. 19 Abnormal pseudouridylation has been implicated in neurological disorders.…”

“…19 Abnormal pseudouridylation has been implicated in neurological disorders. 20 Altered tRNA modifications, including pseudouridylation, have been observed in various cancer types. 9,10,17,18 Nucleic acid function is guided by non-covalent interactions between polynucleotides and with cellular protein machinery.…”