PUS1

PUS1 encodes a multisite-specific pseudouridine synthase that installs pseudouridine in tRNAs across the cytoplasmic, nuclear, and mitochondrial compartments, with structurally distinct isoforms directing dual-compartment localization (PMID:15772074, PMID:17056637). The catalytic mechanism is built on an antiparallel β-sheet fold with a flexible hinge that opens and closes around an electropositive active-site cleft, and two unique C-terminal α-helices form the RNA-binding surface that distinguishes its substrate selectivity from bacterial homologues (PMID:23707380); substrate recognition extends beyond tRNA to mRNA-derived RNA duplexes, where PUS1 engages both strands to guide the target uridine into the active site (PMID:37939088). Catalytic function depends on a structural zinc atom required for native conformation and tRNA binding, and on substrate features such as a G26·A44 base pair that accelerates tRNA association (PMID:9585540, PMID:10356324). Loss of PUS1 catalytic activity through a missense mutation in the catalytic center causes MLASA, abolishing pseudouridylation of mitochondrial and cytoplasmic tRNAs and producing defective mitochondrial translation and combined respiratory chain defects (PMID:15108122, PMID:15772074, PMID:17056637). Beyond canonical tRNA modification, PUS1 stabilizes select mRNAs via pseudouridylation to drive cell migration (PMID:39993614), suppresses intron retention and dsRNA-driven innate immune activation through effects on pre-mRNA splicing (PMID:42003926), acts non-enzymatically to protect EIF3b from proteasomal degradation in metastasis (PMID:39247811), and marks RNAs with pseudouridine for export into extracellular vesicles via the reader protein MYL6 [PMID:bio_10.1101_2025.10.28.685156]. Its expression is transcriptionally controlled by FOXA1 and USF1 in cancer contexts (PMID:39247811, PMID:39993614).

1. 2004 Medium

   Established that PUS1 encodes a pseudouridine synthase whose catalytic activity is essential for mitochondrial function, by linking a catalytic-center mutation to human disease.

   Evidence Linkage analysis, homozygosity mapping, and Sanger sequencing in MLASA families

   PMID:15108122

   Open questions at the time

   • Enzymatic activity not directly tested in this study
   • Mechanistic link between tRNA modification loss and respiratory failure not yet established
2. 2005 High

   Demonstrated directly that the disease mutation abolishes enzymatic activity and tRNA pseudouridylation, and that PUS1 protein distributes across nucleus, cytoplasm, and mitochondria.

   Evidence Pseudouridine and cell-extract enzyme assays in patient-derived lymphoblastoid cells plus immunohistochemistry

   PMID:15772074

   Open questions at the time

   • Mechanism connecting loss of tRNA modification to OXPHOS defect not resolved
   • Targeting signals directing isoforms to compartments not defined
3. 2006 Medium

   Defined the structural basis for dual-compartment targeting, showing the nuclear isoform carries an N-terminal extension absent from the mitochondrial isoform, and tied loss-of-function to reduced mtDNA translation.

   Evidence Isoform sequencing plus respiratory chain assays and mtDNA translation product measurement in patient muscle and fibroblasts

   PMID:17056637

   Open questions at the time

   • Single-lab functional readouts
   • Quantitative contribution of each isoform to compartmental modification not measured
4. 1999 High

   Resolved the enzyme's catalytic and recognition logic, defining it as a multisite-specific synthase acting at tRNA positions 27/28 with a defined kinetic regime and a key recognition determinant.

   Evidence In vitro pseudouridylation with recombinant Pus1p, kinetics, real-time binding, and tRNA variant analysis (yeast ortholog)

   PMID:10356324 PMID:10492022

   Open questions at the time

   • Yeast ortholog rather than human enzyme
   • Catalytic step kinetics distinct from binding not fully dissected
5. 1998 High

   Established that a bound zinc atom plays a structural, not catalytic, role essential for folding and tRNA binding.

   Evidence Atomic absorption, chelation/reconstitution, ultracentrifugation, and spectroscopy with recombinant yeast Pus1p

   PMID:9585540

   Open questions at the time

   • Yeast ortholog; conservation of zinc role in human PUS1 inferred
   • Residues coordinating zinc not mapped to human sequence here
6. 2013 High

   Provided the high-resolution architecture of the human catalytic core, explaining substrate selectivity via unique C-terminal helices and a hinged electropositive cleft.

   Evidence X-ray crystallography of hPus1 catalytic core at 1.8 Å with active-site ligand mimic and tRNA docking

   PMID:23707380

   Open questions at the time

   • No co-crystal with bound tRNA
   • Conformational dynamics of the hinge during catalysis inferred from static structures
7. 2023 High

   Extended substrate scope structurally to mRNA, showing PUS1 binds both strands of an RNA duplex to position the target uridine, diverging from tRNA recognition.

   Evidence X-ray co-crystal structure of yeast PUS1 with an mRNA-derived RNA duplex at 2.4 Å

   PMID:37939088

   Open questions at the time

   • Yeast enzyme; human mRNA recognition mode inferred
   • In vivo prevalence of duplex mRNA substrates not quantified
8. 2024 Medium

   Revealed a non-enzymatic function in which PUS1 stabilizes EIF3b to drive metastasis, decoupling a disease-relevant role from catalytic activity.

   Evidence Enzymatic-dead rescue, Co-IP/protein stability assays, EIF3b rescue, and FOXA1 ChIP/promoter reporter in prostate cancer cells

   PMID:39247811

   Open questions at the time

   • Single lab
   • Direct PUS1–EIF3b binding interface not mapped
   • Generality beyond prostate cancer bone metastasis unknown
9. 2025 Medium

   Identified roles in mRNA stabilization and splicing regulation, showing pseudouridylation stabilizes SMOX mRNA while depletion drives intron retention, dsRNA accumulation, and innate immune activation.

   Evidence Knockdown/overexpression, mRNA stability and pseudouridylation assays, RNA-seq intron retention, dsRNA immunofluorescence, and immune/translation assays in RCC; USF1 ChIP

   PMID:39993614 PMID:42003926

   Open questions at the time

   • Single-lab studies
   • Whether splicing effect is enzymatic or scaffolding not fully resolved
   • Direct targets of pseudouridylation in splicing not enumerated
10. 2025 Medium

    Implicated PUS1-deposited pseudouridine as a sorting signal for RNA export into extracellular vesicles, read by MYL6.

    Evidence Genome-wide CRISPR screen, proteomics, synthetic modified-RNA export assays, and MYL6 binding assays (preprint)

    PMID:bio_10.1101_2025.10.28.685156

    Open questions at the time

    • Preprint, not peer-reviewed
    • MYL6 pseudouridine-reader mechanism not structurally defined
    • Physiological scope of the export pathway unknown

• How PUS1's many enzymatic and non-enzymatic functions are partitioned across compartments, isoforms, and tissues remains unresolved.
• No unified model linking tRNA, mRNA, splicing, EIF3b, and EV-sorting activities
• Isoform-specific function assignment incomplete
• Human in vivo substrate atlas not defined