Affinage

GPN3

GPN-loop GTPase 3 · UniProt Q9UHW5

Length
284 aa
Mass
32.8 kDa
Annotated
2026-06-10
18 papers in source corpus 15 papers cited in narrative 15 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

GPN3 is a conserved GPN-loop GTPase that functions as a dedicated assembly and nuclear-import chaperone for RNA polymerase II, acting as part of a broader machinery required for transcription and cell proliferation (PMID:21768307, PMID:21782856, PMID:31431135). It stably associates with RNAPII and directly contacts the RPB7/RPB4 subunits and the CTD; loss of GPN3 causes cytoplasmic retention of the largest subunit Rpb1 and a corresponding drop in RNA synthesis (PMID:21768307, PMID:21782856). Mechanistically, GPN3 forms a tight, mutually stabilizing heterodimer with GPN1 (yeast Npa3) through an interface built from α-helix 7, insertion 2, and the GPN-loop, and disruption of this interface abolishes the proteins' function in RNAPII localization (PMID:25241168, PMID:31298811, PMID:32985767). The GPN3/GPN1 module participates directly in folding and assembly of the two largest RNAPII subunits (Rpb1, Rpb2) together with the phosphatase Rtr1, such that when GPN3 is defective, RNAPII subunits coalesce into reversible, protein-based cytoplasmic condensates (the RNAPII Assembly Stress Response) that recruit the chaperone Hsp82 and trigger transcriptional reprogramming of ribosome-biogenesis and metabolic genes (PMID:35314265, PMID:36190433, PMID:41500282). GPN3 activity extends to other polymerases, directing RNA polymerase III localization in yeast and, downstream of MYC, sensing GTP sufficiency to target RNA polymerase I to ribosomal DNA (PMID:23267056, PMID:33079728). GPN3 levels and location are tightly controlled: it shuttles via CRM1-dependent nuclear export under the influence of serum and cell density, and is polyubiquitinated on K216 and degraded by the proteasome specifically in the nucleus, a degradation that GPN1 binding suppresses (PMID:29029378, PMID:38342311). A cancer-associated Q279* nonsense mutation creates a PDZ-binding motif that drives GPN3 into the nucleus, blocks GPN1 nuclear export, and dominantly impairs RNAPII accumulation and transcription (PMID:28940195). Beyond transcription, GPN3 interacts with clathrin machinery (CLTA, AP2B1, AP2S1) and EGFR to regulate clathrin-mediated endocytosis and prolong EGFR signaling in a GTP-dependent manner (PMID:39893205).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 2011 High

    Established the founding mechanistic role of GPN3 by showing it is a physical partner of RNAPII required for the polymerase's nuclear accumulation, answering whether GPN3 acts in RNAPII biogenesis.

    Evidence Reciprocal Co-IP, siRNA/RNAi knockdown with subcellular fractionation and RNA synthesis assays in human and MCF-12A cells

    PMID:21768307 PMID:21782856

    Open questions at the time
    • Did not resolve whether the defect is in import per se or upstream assembly/folding
    • Direct subunit contacts beyond RPB7/RPB4 and CTD not mapped structurally
  2. 2012 High

    Placed GPN3 in a conserved genetic pathway and broadened its scope to RNAPIII, showing GPN proteins act upstream of Iwr1 and that their defect is not bypassed by adding an NLS to a subunit.

    Evidence Temperature-sensitive alleles, genetic epistasis and NLS-Rpb3 fusion suppression in S. cerevisiae

    PMID:23267056

    Open questions at the time
    • Biochemical step catalyzed by GPN3 within the pathway not defined
    • Mechanism distinguishing GPN function from simple nuclear-import defect unresolved
  3. 2014 High

    Defined GPN3 as an obligate heterodimer partner of GPN1, answering how the two GTPases are organized and showing they co-stabilize and co-shuttle.

    Evidence Reciprocal Co-IP of endogenous proteins, leptomycin B export inhibition, tagged co-localization in mammalian cells

    PMID:25241168

    Open questions at the time
    • Interface residues and stoichiometry not yet defined
    • Functional consequence of shuttling not established
  4. 2017 High

    Revealed nuclear-specific quality control of GPN3, identifying K216 polyubiquitination and proteasomal degradation in the nucleus and GPN1 as a protective factor.

    Evidence MG132 inhibition, K216R/K189 mutagenesis, pulse-chase half-life, GPN1 co-expression and fractionation

    PMID:29029378

    Open questions at the time
    • E3 ligase responsible for K216 ubiquitination not identified
    • Physiological signal triggering nuclear degradation unknown
  5. 2017 Medium

    Connected GPN3 to cancer by showing a Q279* nonsense mutation generates a PDZ-binding motif that dominantly disrupts GPN1 export and RNAPII accumulation.

    Evidence RNAi replacement with resistant constructs, PDZ-motif mutagenesis, localization and transcription assays

    PMID:28940195

    Open questions at the time
    • PDZ-domain partner mediating the effect not identified
    • Prevalence and oncogenic relevance of Q279* in patients not established
  6. 2019 High

    Mapped the structural basis of the GPN1-GPN3 heterodimer and proved the interface is functionally required, answering how the two GTPases assemble.

    Evidence Live-cell FRET, modeling on the Npa3 crystal structure, interface mutagenesis (W132D, M227D) and RNAPII localization readout

    PMID:31298811

    Open questions at the time
    • No experimental high-resolution structure of the human heterodimer
    • GTP hydrolysis cycle coupling to heterodimer assembly not defined
  7. 2019 Medium

    Linked GPN3's RNAPII-targeting function to proliferation in disease cells, showing knockdown impairs growth across breast cancer subtypes.

    Evidence shRNA knockdown, Rpb1 localization, proliferation and mammosphere assays in multiple breast cancer lines

    PMID:31431135

    Open questions at the time
    • Whether proliferation defect is solely via RNAPII or other pathways untested
    • In vivo tumor relevance not assessed
  8. 2020 High

    Demonstrated by rapid depletion that GPN3 is required acutely for RNAPII assembly and confirmed conserved mutual dependency with Npa3/GPN1 in yeast and human.

    Evidence Auxin-inducible degron depletion, Co-IP, RNAPII subunit localization, multicopy suppressor screen

    PMID:32985767

    Open questions at the time
    • Direct biochemical assembly intermediate not isolated
    • Order of subunit incorporation relative to GPN3 action unclear
  9. 2021 Medium

    Extended GPN3 function to RNA polymerase I and nutrient sensing, showing MYC upregulates GPN1/3 and that constitutively GTP-bound mutants rescue Pol I targeting under GTP depletion.

    Evidence Coexpression analysis, constitutively GTP-bound mutants, IMPDH inhibition, Pol I rDNA localization

    PMID:33079728

    Open questions at the time
    • Direct binding of GPN3 to Pol I subunits not shown
    • How GTP-binding state is sensed mechanistically not resolved
  10. 2022 Medium

    Defined GPN3's biochemical role as direct participation in assembling Rpb1 and Rpb2 and characterized the cytoplasmic foci phenotype as a reversible assembly stress response that also depends on Rtr1.

    Evidence Temperature-sensitive alleles, tagged-subunit microscopy, multicopy RTR1 suppression, catalytic-mutant and reversibility experiments

    PMID:35314265 PMID:36190433

    Open questions at the time
    • Precise enzymatic contribution of GPN3 versus Rtr1 in assembly not separated
    • Composition of the assembly intermediate not defined
  11. 2024 Medium

    Resolved how GPN3 localization is controlled, identifying CRM1-dependent NES motifs and serum/cell-density regulation, and showed nuclear export is needed for proliferation.

    Evidence Leptomycin B, MG132, NES mutagenesis, proliferation assay, live-cell localization under varied serum/density

    PMID:38342311

    Open questions at the time
    • Signaling pathway coupling serum/density to shuttling not mapped
    • Cargo/adaptor recognizing GPN3 NES motifs not identified
  12. 2025 Medium

    Uncovered a transcription-independent role for GPN3 in clathrin-mediated endocytosis and EGFR signaling, dependent on GTP abundance.

    Evidence Co-IP with CLTA/AP2B1/AP2S1/EGFR, overexpression/knockdown, endocytosis and clathrin-coated pit assays, co-localization microscopy

    PMID:39893205

    Open questions at the time
    • Direct versus indirect nature of GPN3-clathrin interactions not fully resolved
    • Whether endocytic and RNAPII roles share a common GTPase mechanism unknown
  13. 2026 Medium

    Characterized the biophysical nature of the RNAPII Assembly Stress Response, showing GPN3 inactivation produces protein-based dynamic condensates that recruit Hsp82 and reprogram ribosome-biogenesis and metabolic transcription.

    Evidence GPN inactivation, FRAP, 1,6-hexanediol and nuclease treatments, transcriptomics, Hsp82 co-localization

    PMID:41500282

    Open questions at the time
    • Whether condensate formation is protective or deleterious not resolved
    • Mechanism linking RASR to transcriptional reprogramming not defined

Open questions

Synthesis pass · forward-looking unresolved questions
  • How GPN3's GTP-binding/hydrolysis cycle is mechanistically coupled to RNAPII subunit folding, heterodimer assembly with GPN1, and its distinct endocytic role remains unresolved.
  • No reconstituted in vitro assembly assay defining GPN3 GTPase enzymology
  • GTPase-activating and nucleotide-exchange factors for GPN3 unidentified
  • No experimental structure of GPN3 bound to RNAPII subunits

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003924 GTPase activity 4 GO:0044183 protein folding chaperone 2 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005634 nucleus 3 GO:0005829 cytosol 3 GO:0005730 nucleolus 1
Pathway
R-HSA-74160 Gene expression (Transcription) 3 R-HSA-392499 Metabolism of proteins 2 R-HSA-9609507 Protein localization 2 R-HSA-5653656 Vesicle-mediated transport 1
Partners
AP2B1AP2S1CLTAEGFRGPN1RPB4RPB7RTR1
Complex memberships
GPN1-GPN3 heterodimer

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2011 Human GPN3 stably associates with RNA polymerase II (RNAPII) in both cytoplasmic and nuclear fractions, directly interacts with RNAPII subunit RPB7/RPB4 and the CTD of RNAPII, and depletion of GPN3 by siRNA causes decreased RNAPII levels in the nucleus with cytoplasmic accumulation, establishing GPN3's role in nuclear import of RNAPII. Co-immunoprecipitation, siRNA knockdown, subcellular fractionation, dominant-negative GTP-binding pocket mutant stable cell lines Molecular and cellular biology High 21768307
2011 Parcs/Gpn3 co-immunoprecipitates with RNA polymerase II, and its knockdown by RNAi causes cytoplasmic retention of Rpb1 (largest RNAPII subunit) and reduction in overall RNA synthesis in MCF-12A cells, demonstrating a critical role for Gpn3 in nuclear accumulation of RNAPII and transcription. RNAi knockdown, co-immunoprecipitation, subcellular localization by immunofluorescence, RNA synthesis assay Biochimica et biophysica acta High 21782856
2012 In S. cerevisiae, temperature-sensitive alleles of GPN3 cause RNAPII nuclear localization defects and hypersensitivity to transcription inhibition; GPN3 mutants also exhibit RNA polymerase III localization defects. Genetic epistasis shows GPN proteins function upstream of Iwr1 in RNAPII/III biogenesis, as the iwr1Δ nuclear import defect is partially suppressed by NLS-Rpb3 fusion whereas GPN3 mutant defects are not. Temperature-sensitive alleles, genetic epistasis, nuclear localization assays, NLS fusion suppression experiments Genetics High 23267056
2014 Gpn1 and Gpn3 associate tightly as a complex in mammalian cells: all endogenous Gpn3 co-immunoprecipitates with Gpn1-Flag and vice versa. Gpn1-Gpn3 interaction maintains steady-state protein levels of both GTPases, and the complex undergoes nucleocytoplasmic shuttling revealed by leptomycin B treatment. Co-immunoprecipitation, leptomycin B nuclear export inhibition, EYFP/Flag-tagged co-expression localization FEBS letters High 25241168
2017 Gpn3 is polyubiquitinated on lysine 216 (but not K189) and degraded by the proteasome specifically in the cell nucleus. Gpn3-Flag undergoes nucleocytoplasmic shuttling, but polyubiquitination and proteasomal degradation occur only in the nucleus. Gpn1 inhibits Gpn3 polyubiquitination in a dose-dependent manner, protecting Gpn3 from degradation. Proteasome inhibition (MG132), site-directed mutagenesis (K216R), pulse-chase half-life assay, co-expression with Gpn1-EYFP, subcellular fractionation FEBS letters High 29029378
2017 A cancer-associated Q279* nonsense mutation in Gpn3 generates a PDZ-binding motif that causes Gpn3 to enter the nucleus and inhibit Gpn1 nuclear export, resulting in markedly decreased RNAPII nuclear accumulation and transcriptional activity. The dominant effect requires the PDZ-binding motif generated by the Q279* mutation. RNAi replacement with RNAi-resistant constructs, subcellular localization, transcriptional activity assay, PDZ-binding motif mutagenesis FEBS letters Medium 28940195
2019 FRET analysis and molecular modeling reveal that human Gpn1 and Gpn3 associate through a large heterodimer interface formed by internal α-helix 7, insertion 2, and the GPN-loop from each protein. W132D and M227D mutations in Gpn1 disrupt interaction with Gpn3 by FRET and abolish the dominant-negative effect on RNAPII localization, demonstrating that an intact Gpn1-Gpn3 interaction is required for their cellular function. FRET (live cell), molecular modeling based on Npa3 crystal structure, site-directed mutagenesis of interface residues, RNAPII localization assay The FEBS journal High 31298811
2019 shRNA-mediated Gpn3 knockdown in breast cancer cell lines causes cytoplasmic retention of a fraction of Rpb1 and markedly decreases cell proliferation across multiple breast cancer subtypes regardless of transformation level, confirming Gpn3 is required for RNAPII nuclear targeting and cell proliferation in breast cancer cells. shRNA knockdown, subcellular localization of Rpb1, cell proliferation assay, mammosphere assay Technology in cancer research & treatment Medium 31431135
2020 In yeast, rapid degradation of Gpn3 (using auxin-inducible degron system) leads to cytoplasmic accumulation of Rpb1 and defects in RNAPII assembly. Npa3/Gpn1 physically interacts with Gpn3, and there is a mutual dependency of Npa3 and Gpn3 protein levels. Human Gpn1 also physically interacts with Gpn3, paralleling the yeast interaction. Auxin-inducible degron (AID) system, co-immunoprecipitation, RNAPII subunit localization assay, multicopy genetic suppressor screening FASEB journal High 32985767
2021 GPN1 and GPN3 are upregulated by MYC and direct RNA polymerase I (Pol I) to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigate the effect of GTP depletion on Pol I localization, demonstrating that GPN1/3 function as GTP-sensing molecules that link nucleotide sufficiency to ribosome biogenesis. Coexpression analysis, constitutively GTP-bound mutants, IMPDH inhibition (GTP depletion), Pol I localization to rDNA assay The Journal of clinical investigation Medium 33079728
2022 Gpn3 and Npa3 directly participate in assembly of the two largest RNAPII subunits (Rpb1 and Rpb2). When Gpn3 is defective, RNAPII assembly is disrupted and RNAPII subunits accumulate as cytoplasmic foci (termed 'RNAPII assembly stress response'), which is reversible upon recovery of the assembly factor. Temperature-sensitive gpn3 mutant alleles, fluorescence microscopy of tagged RNAPII subunits, genetic suppression, reversibility experiments International journal of biological macromolecules Medium 35314265
2022 Rtr1 cooperates with Gpn3 and Npa3 to assemble RNAPII; multiple copies of RTR1 suppress cytoplasmic clumping of RNAPII subunits in gpn3-9 mutant. Deletion of RTR1 leads to cytoplasmic clumping of RNAPII subunits, placing Rtr1 in the same assembly pathway as Gpn3. Multicopy genetic suppression, RTR1 deletion, fluorescence microscopy of RNAPII subunit localization, catalytically inactive Rtr1 mutant FASEB journal Medium 36190433
2024 Gpn3 nucleocytoplasmic shuttling is regulated by CRM1-mediated nuclear export (sensitive to leptomycin B) and by proteasomal degradation. Five NES motifs were identified in Gpn3 primary sequence; inactivation of NES1 or NES3 has the most robust effect on nuclear accumulation. Cells expressing exclusively NES-deficient Gpn3 proliferate slower, indicating nuclear export is important for Gpn3 function. Cell density and serum (growth factors) regulate Gpn3 shuttling: serum stimulation causes rapid but transient nuclear accumulation. Leptomycin B treatment, MG132 proteasome inhibition, NES mutagenesis, cell proliferation assay, live-cell fluorescence localization under varying cell density and serum conditions Biochimica et biophysica acta. Molecular cell research Medium 38342311
2025 GPN3 interacts with clathrin light chain A (CLTA), AP2B1, and AP2S1. Upregulation of GPN3 inhibits clathrin-coated pit invagination. GPN3 interacts with EGFR and regulates co-localization of EGFR and CLTA as well as EGFR localization in early endosomes upon EGF stimulation, leading to decreased endocytic levels of EGFR and increased EGFR membrane accumulation and prolonged EGFR signaling activation. These effects are dependent on cellular GTP abundance. Co-immunoprecipitation (GPN3 with CLTA, AP2B1, AP2S1, EGFR), overexpression and knockdown, EGFR endocytosis assay, clathrin-coated pit assay, co-localization microscopy Cell death discovery Medium 39893205
2026 Inactivation of Gpn3 (along with other GPN proteins) results in reversible accumulation of Rpb1, Rpb2, and Rpb3 in cytoplasmic foci (RNAPII Assembly Stress Response, RASR). The molecular chaperone Hsp82 accumulates in and partially co-localizes with these foci. The condensates are protein-based, nucleic acid-free, resist hexanediol dissolution, and show dynamic behavior by FRAP. RASR triggers coordinated transcriptional reprogramming of ribosome biogenesis genes and metabolic pathways. GPN protein inactivation, fluorescence microscopy, FRAP, 1,6-hexanediol treatment, RNase/DNase treatment, transcriptomic profiling, Hsp82 co-localization International journal of biological macromolecules Medium 41500282

Source papers

Stage 0 corpus · 18 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2021 Guanosine triphosphate links MYC-dependent metabolic and ribosome programs in small-cell lung cancer. The Journal of clinical investigation 62 33079728
2011 Human GTPases associate with RNA polymerase II to mediate its nuclear import. Molecular and cellular biology 51 21768307
2012 Biogenesis of RNA polymerases II and III requires the conserved GPN small GTPases in Saccharomyces cerevisiae. Genetics 42 23267056
2011 Parcs/Gpn3 is required for the nuclear accumulation of RNA polymerase II. Biochimica et biophysica acta 35 21782856
2020 Npa3 interacts with Gpn3 and assembly factor Rba50 for RNA polymerase II biogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 15 32985767
2014 Gpn1 and Gpn3 associate tightly and their protein levels are mutually dependent in mammalian cells. FEBS letters 12 25241168
2019 FRET-based analysis and molecular modeling of the human GPN-loop GTPases 1 and 3 heterodimer unveils a dominant-negative protein complex. The FEBS journal 10 31298811
2019 Gpn3 Is Essential for Cell Proliferation of Breast Cancer Cells Independent of Their Malignancy Degree. Technology in cancer research & treatment 7 31431135
2022 Npa3-Gpn3 cooperate to assemble RNA polymerase II and prevent clump of its subunits in the cytoplasm. International journal of biological macromolecules 6 35314265
2022 Rtr1 is required for Rpb1-Rpb2 assembly of RNAPII and prevents their cytoplasmic clump formation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 6 36190433
2017 The Gpn3 Q279* cancer-associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting. FEBS letters 5 28940195
2018 A rAAV2-producing yeast screening model to identify host proteins enhancing rAAV DNA replication and vector yield. Biotechnology progress 4 30298993
2025 GTPase GPN3 facilitates cell proliferation and migration in non-small cell lung cancer by impeding clathrin-mediated endocytosis of EGFR. Cell death discovery 3 39893205
2017 How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context? Molecular bioSystems 2 28352859
2017 Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1-inhibitable manner. FEBS letters 2 29029378
2024 Nucleocytoplasmic shuttling of the GPN-loop GTPase Gpn3 is regulated by serum and cell density in MCF-12A mammary cells. Biochimica et biophysica acta. Molecular cell research 1 38342311
2026 Reversible cytoplasmic foci of RNA polymerase II subunits serve as proteostatic hubs orchestrating transcriptional reprogramming. International journal of biological macromolecules 0 41500282
2025 Proposal of Chemical Inhibitors That Compete with the Binding of RNA Polymerase II Subunits to Essential GTPases GPN Npa3 and Gpn1. ACS omega 0 41244487

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