Affinage

PHAX

Phosphorylated adapter RNA export protein · UniProt Q9H814

Length
394 aa
Mass
44.4 kDa
Annotated
2026-04-28
13 papers in source corpus 10 papers cited in narrative 10 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

PHAX is a phosphorylation-regulated adaptor protein that scaffolds the nuclear export of U snRNAs and mediates intranuclear routing of small non-coding RNAs. It bridges the cap-binding complex (CBC) on capped RNA to the CRM1/RanGTP export receptor through its central region, with phosphorylation in the nucleus required for export complex assembly and cytoplasmic dephosphorylation driving disassembly to ensure directional transport (PMID:10786834). PHAX contains a novel RNA-binding domain (RNA_GG_bind) that is intrinsically disordered and folds upon binding single-stranded RNA, two nuclear localization signals for reimport, and a separate CBC-interaction domain (PMID:11333016, PMID:20430857). Beyond snRNA export, PHAX transports box C/D snoRNA precursors (U3, U8, U13) and telomerase RNA to Cajal bodies, facilitates nuclear export of the short H2AX mRNA, and its loading onto CBC-bound RNA is regulated by competition with hnRNP C on longer transcripts and stimulated by the TREX component UAP56/DDX39B via ALYREF bridging (PMID:15574332, PMID:32759388, PMID:36620872, PMID:39011894).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 2000 High

    Identification of PHAX as the missing adaptor that explained how U snRNAs are selectively loaded onto CRM1 for nuclear export, establishing that a phosphorylation-dephosphorylation cycle across compartments provides directionality.

    Evidence In vitro export complex reconstitution, in vivo depletion, mass spectrometry identification from HeLa cell extracts

    PMID:10786834

    Open questions at the time
    • Kinase(s) and phosphatase(s) responsible for the compartmentalized phosphorylation cycle were not identified
    • Structural basis for how phosphorylation promotes complex assembly was unknown
  2. 2001 High

    Domain mapping revealed PHAX acts as a modular scaffold with separable RNA-binding, CBC-interaction, and NLS domains, explaining how it can simultaneously contact RNA, CBC, and recycle back to the nucleus after export.

    Evidence Systematic deletion/mutagenesis with in vitro binding and nuclear export assays

    PMID:11333016

    Open questions at the time
    • Three-dimensional structure of the RNA-binding domain was not yet resolved
    • Whether PHAX recognizes RNA with sequence specificity was unresolved
  3. 2004 High

    Discovery that PHAX functions beyond snRNA export by mediating intranuclear transport of box C/D snoRNA precursors and telomerase RNA to Cajal bodies broadened its role to a general small RNA identity factor.

    Evidence RNA immunoprecipitation, PHAX/CRM1 inactivation, fluorescence microscopy of RNA localization in HeLa cells

    PMID:15574332

    Open questions at the time
    • How PHAX discriminates between substrates destined for Cajal bodies versus nuclear export was unclear
    • Whether Cajal body targeting requires the same phosphorylation cycle was not tested
  4. 2010 High

    Structural characterization revealed the RNA_GG_bind domain undergoes a disorder-to-order transition upon RNA binding, explaining how PHAX achieves sequence-nonspecific recognition of single-stranded RNA substrates.

    Evidence NMR spectroscopy, X-ray crystallography, mutagenesis with functional export readout

    PMID:20430857

    Open questions at the time
    • Structure of full-length PHAX in complex with CBC and CRM1 was unknown
    • Basis for length-dependent selectivity of PHAX substrates was not addressed
  5. 2020 Medium

    Demonstration that PHAX promotes export and expression of the short H2AX mRNA extended its substrate range to mRNAs and connected PHAX to the DNA damage response.

    Evidence siRNA knockdown, RT-qPCR, γH2AX immunostaining, DNA damage sensitivity assays in human cells

    PMID:32759388

    Open questions at the time
    • The dual effect on H2AX transcription and export was not mechanistically separated
    • Whether other short mRNAs are similarly PHAX-dependent was not surveyed systematically
  6. 2023 Medium

    Identification of hnRNP C tetramer as a competitive inhibitor of PHAX recruitment to CBC on longer RNAs explained the transcript-length checkpoint that sorts short snRNAs from long mRNAs into distinct export pathways.

    Evidence Co-immunoprecipitation, in vitro binding assays, hnRNP C tetramerization mutants, RNA-length-dependent export assays

    PMID:36620872

    Open questions at the time
    • Precise RNA length threshold at which hnRNP C outcompetes PHAX was not defined
    • Whether hnRNP C competition also regulates PHAX intranuclear routing functions was not tested
  7. 2024 Medium

    Discovery that TREX components UAP56/DDX39B stimulate PHAX loading onto RNA in an ATP-dependent manner, with ALYREF bridging, revealed an active remodeling step upstream of export complex formation.

    Evidence In vitro reconstitution with ATP-dependent factors, co-immunoprecipitation, in vivo knockdown in human cells

    PMID:39011894

    Open questions at the time
    • Whether UAP56 directly remodels RNA structure to facilitate PHAX binding or acts allosterically on PHAX was not resolved
    • Relationship between TREX-mediated PHAX loading and the hnRNP C checkpoint was not integrated
  8. 2024 High

    Cryo-EM structure of the complete snRNA export complex revealed how phosphorylated PHAX bridges CBC-capped RNA to CRM1-RanGTP through synergistic contacts and displaces ARS2, committing the ribonucleoprotein to export.

    Evidence Cryo-EM structure determination, in vitro and in-cell mutagenesis (preprint)

    PMID:bio_10.1101_2024.11.28.625805

    Open questions at the time
    • Preprint awaiting peer review
    • Specific phosphorylation sites making RanGTP contacts were identified structurally but their individual functional contributions were not fully dissected
    • Structural basis for PHAX interaction with snoRNAs in the Cajal body pathway was not addressed

Open questions

Synthesis pass · forward-looking unresolved questions
  • The kinase(s) and phosphatase(s) governing the PHAX phosphorylation cycle remain unidentified, and how PHAX differentially engages its snRNA export versus intranuclear snoRNA routing functions at the structural level is unresolved.
  • No kinase or phosphatase has been assigned to the PHAX phosphorylation cycle
  • Whether PHAX uses the same or distinct surfaces for Cajal body targeting versus nuclear export is unknown
  • Genome-wide identification of PHAX RNA substrates (e.g., by CLIP) has not been reported

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 3 GO:0003723 RNA binding 2
Localization
GO:0005634 nucleus 3 GO:0005829 cytosol 1
Pathway
R-HSA-8953854 Metabolism of RNA 3
Partners
ALYREFDDX39BHNRNPCLIN28BNCBP1NCBP2RANXPO1
Complex memberships
snRNA export complex (PHAX-CBC-CRM1-RanGTP)

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2000 PHAX (phosphorylated adaptor for RNA export) is an essential adaptor for U snRNA nuclear export, assembling into an export complex with CBC, CRM1/Xpo1, and RanGTP. PHAX is phosphorylated in the nucleus (required for export complex assembly) and dephosphorylated in the cytoplasm (causing export complex disassembly), with this compartmentalized phosphorylation cycle contributing to directionality of export. In vitro export complex assembly assay, in vivo knockdown/depletion, biochemical fractionation, mass spectrometry identification Cell High 10786834
2001 The most evolutionarily conserved region of PHAX constitutes a novel RNA-binding domain (RNA_GG_bind domain) essential for U snRNA export; PHAX also contains two major nuclear localization signals (NLSs) required for nuclear recycling after export, and a separate domain mediating interaction with CBC. PHAX acts as a scaffold for assembly of U snRNA export complexes. Systematic domain deletion/mutagenesis analysis, in vitro binding assays, nuclear export assays RNA (New York, N.Y.) High 11333016
2004 PHAX binds m7G-capped U3 snoRNA precursors and is required for their transport to Cajal bodies; subsequently CRM1 routes U3 from Cajal bodies to nucleoli. PHAX also binds precursors of U8, U13 box C/D snoRNAs, and telomerase RNA, indicating PHAX plays a broad role in determining RNA identity for intranuclear transport beyond snRNA export. RNA immunoprecipitation, co-immunoprecipitation, PHAX and CRM1 inactivation (dominant-negative/RNAi), fluorescence microscopy of RNA localization Molecular cell High 15574332
2010 The PHAX RNA-binding domain (RNA_GG_bind domain) adopts a novel helical fold that is intrinsically disordered as a monomer and folds upon RNA binding; it binds single-stranded RNA with micromolar affinity without sequence specificity. Mutational analysis confirmed that RNA-binding by this domain is essential for PHAX-mediated nuclear export. NMR spectroscopy, X-ray crystallography, RNA binding assays, mutagenesis coupled to nuclear export functional assay RNA (New York, N.Y.) High 20430857
2020 PHAX is required for efficient DNA damage response (DDR) by regulating H2AX expression: PHAX knockdown reduces H2AX mRNA levels by inhibiting both transcription of the H2AX gene and nuclear export of H2AX mRNA (one of the shortest mRNAs), leading to reduced γH2AX and increased DNA damage sensitivity. siRNA knockdown, RT-qPCR, nuclear export assays, γH2AX immunostaining, DNA damage sensitivity assays RNA (New York, N.Y.) Medium 32759388
2023 The hnRNP C tetramer binds CBC on mRNA and impedes PHAX recruitment to longer RNA polymerase II transcripts, routing them to the mRNA export pathway rather than the snRNA export pathway. The tetramer-forming activity and strong RNA-binding of hnRNP C are critical for blocking PHAX access to CBC on longer RNAs. Co-immunoprecipitation, in vitro binding assays, mutagenesis of hnRNP C tetramerization domain, RNA-length-dependent export assays Nucleic acids research Medium 36620872
2024 The RNA helicase UAP56/DDX39B (and its paralog URH49/DDX39A), as components of the TREX complex, stimulate RNA binding of PHAX in an ATP-dependent manner to promote U snRNA export. ALYREF acts as a bridge between PHAX and UAP56/DDX39B. This TREX-mediated loading of PHAX onto RNA is mechanistically distinct from mRNA export. In vitro reconstitution of U snRNA export with ATP-dependent factors, identification by biochemical fractionation, co-immunoprecipitation, in vivo knockdown assays Nucleic acids research Medium 39011894
2024 Cryo-EM structure of the complete snRNA export complex (phosphorylated PHAX + CBC + CRM1 + RanGTP + capped RNA) reveals that the central region of PHAX bridges CBC-bound capped RNA to CRM1-RanGTP while reinforcing cap dinucleotide binding; phosphorylated regions of PHAX make essential contacts with the basic surface of RanGTP and a distant region of CRM1. Formation of this complex displaces ARS2 from CBC and is incompatible with CBC interactions with ALYREF or NCBP3, committing the complex to export. Synergistic binding of all components is required for complex assembly. Cryo-EM structure determination, in vitro mutagenesis binding/export assays, in-cell mutagenesis functional assays bioRxiv (preprint)preprint High bio_10.1101_2024.11.28.625805
2024 In glioma cells, PHAX is activated by HRasV12 signaling and recruits U3 snoRNAs, which in turn recruit DNA-PKcs in a Ku-dependent manner; overexpressed TRIM24 is also recruited by PHAX to U3 snoRNAs, enabling DNA-PKcs to phosphorylate TRIM24 at S767/768 residues, promoting epigenome reprogramming. Co-immunoprecipitation, knockdown experiments, phospho-site mutagenesis, single-cell RNA-seq Advanced science Low 38828688
2024 PHAX directly binds LIN28B and enhances LIN28B-mediated stabilization of PBX3 mRNA in esophageal cancer cells, representing a non-export RNA regulatory function of PHAX. Co-immunoprecipitation, RNA immunoprecipitation, knockdown with mRNA stability assays, in vivo tumor xenograft Cancer science Low 39668567

Source papers

Stage 0 corpus · 13 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2000 PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation. Cell 291 10786834
2004 PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli. Molecular cell 144 15574332
2001 The evolutionarily conserved region of the U snRNA export mediator PHAX is a novel RNA-binding domain that is essential for U snRNA export. RNA (New York, N.Y.) 37 11333016
2014 A syndromic form of Pierre Robin sequence is caused by 5q23 deletions encompassing FBN2 and PHAX. European journal of medical genetics 20 25195018
2010 Structure and RNA recognition by the snRNA and snoRNA transport factor PHAX. RNA (New York, N.Y.) 13 20430857
2023 The hnRNP C tetramer binds to CBC on mRNA and impedes PHAX recruitment for the classification of RNA polymerase II transcripts. Nucleic acids research 12 36620872
2024 TRIM24 Cooperates with Ras Mutation to Drive Glioma Progression through snoRNA Recruitment of PHAX and DNA-PKcs. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 9 38828688
2020 The RNA transport factor PHAX is required for proper histone H2AX expression and DNA damage response. RNA (New York, N.Y.) 8 32759388
2024 The RNA helicase DDX39 contributes to the nuclear export of spliceosomal U snRNA by loading of PHAX onto RNA. Nucleic acids research 6 39011894
2019 Elevated levels of autoantibodies against EXD2 and PHAX in the sera of patients with chronic thromboembolic pulmonary hypertension. PloS one 5 30759165
2022 Allele-specific alternative splicing of Drosophila Ribosomal protein S21 suppresses a lethal mutation in the Phosphorylated adaptor for RNA export (Phax) gene. G3 (Bethesda, Md.) 4 35920767
2024 PHAX enhanced LIN28B-mediated PBX3 mRNA stability to promote esophageal cancer development. Cancer science 3 39668567
2020 The Efficacy of Sunitinib Treatment of Renal Cancer Cells Is Associated with the Protein PHAX In Vitro. Biology 3 32272660