{"gene":"PHAX","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2000,"finding":"PHAX (phosphorylated adaptor for RNA export) is an essential factor for U snRNA nuclear export complex assembly in vitro; it directly contacts RNA, the nuclear cap-binding complex (CBC), and the export receptor CRM1/Xpo1, and requires phosphorylation for export complex assembly. Dephosphorylation in the cytoplasm causes export complex disassembly, providing directionality.","method":"In vitro export complex assembly assay, in vivo knockdown/depletion, identification of phosphorylation state by biochemistry","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstituted in vitro export complex, in vivo depletion with specific phenotype, multiple orthogonal methods in a single rigorous study","pmids":["10786834"],"is_preprint":false},{"year":2001,"finding":"The most evolutionarily conserved region of PHAX is a novel RNA-binding domain (RBD) essential for U snRNA export. PHAX also contains two major nuclear localization signals (NLSs) required for nuclear recycling after export, and a CBC interaction domain at least partly distinct from the RBD and NLSs, allowing PHAX to act as a scaffold for U snRNA export complex assembly.","method":"Systematic mutagenesis/deletion analysis of PHAX domains combined with in vivo export assays and binding assays","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — domain mutagenesis with functional in vivo and in vitro validation, multiple orthogonal methods","pmids":["11333016"],"is_preprint":false},{"year":2004,"finding":"PHAX binds m7G-capped U3 snoRNA precursors (as well as U8, U13 box C/D snoRNA precursors and telomerase RNA) and is required for transport of U3 snoRNA to Cajal bodies in the first step of intranuclear routing; CRM1 is subsequently required for routing from Cajal bodies to nucleoli. PHAX does not export m7G-capped U3 because its cap becomes hypermethylated in the nucleus.","method":"Immunoprecipitation of RNA–protein complexes, inactivation of PHAX and CRM1 in vivo, fluorescence microscopy","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP/RIP combined with in vivo inactivation and microscopy, single lab but multiple orthogonal methods","pmids":["15574332"],"is_preprint":false},{"year":2010,"finding":"The PHAX RNA-binding domain (RNA_GG_bind domain) adopts a novel helical fold and is monomeric in the absence of ligand, only adopting a tertiary structure upon RNA binding; it binds single-stranded RNA with micromolar affinity without sequence specificity. Mutational analysis confirmed that RNA-binding by PHAX-RBD is required for PHAX-mediated nuclear export.","method":"NMR spectroscopy, X-ray crystallography, mutational analysis of RNA binding","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR and crystal structure combined with mutagenesis and functional export assay, single lab","pmids":["20430857"],"is_preprint":false},{"year":2020,"finding":"PHAX is required for efficient DNA damage response (DDR): 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 cellular sensitivity to DNA damage.","method":"siRNA knockdown of PHAX, RT-qPCR/Northern blot for H2AX mRNA, γH2AX immunofluorescence, DNA damage sensitivity assays","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotype and mRNA-level mechanistic readout, single lab, multiple readouts","pmids":["32759388"],"is_preprint":false},{"year":2023,"finding":"The CBC directly interacts with hnRNP C on mRNA, and the tetramer-forming activity and strong RNA-binding activity of hnRNP C together impede PHAX recruitment to longer transcripts, thereby directing them to the mRNA export pathway rather than the U snRNA export pathway. This reveals the molecular mechanism by which length-dependent RNA classification excludes PHAX from long Pol II transcripts.","method":"Co-immunoprecipitation of CBC–hnRNP C complex on mRNA, RNA-binding and tetramerization mutants of hnRNP C, in vivo export assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with mutational dissection, single lab, multiple orthogonal assays","pmids":["36620872"],"is_preprint":false},{"year":2024,"finding":"The RNA helicase UAP56/DDX39B (and its paralog URH49/DDX39A) stimulates RNA binding of PHAX in an ATP-dependent manner, thereby loading PHAX onto U snRNA and promoting U snRNA export. ALYREF acts as a bridge between PHAX and UAP56/DDX39B. This TREX-component-dependent mechanism for U snRNA export is distinct from the mRNA export mechanism.","method":"In vitro ATP-dependent factor identification assay, RNA-binding assays, co-immunoprecipitation, in vivo U snRNA export assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — novel in vitro reconstitution strategy plus co-IP and in vivo functional validation, single lab, multiple orthogonal methods","pmids":["39011894"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of the complete snRNA export complex (phosphorylated PHAX + CBC + CRM1 + Ran-GTP + capped RNA) reveals that the central region of PHAX bridges CBC-bound capped RNA to CRM1–RanGTP, reinforces cap dinucleotide binding, and contacts CRM1 via a phosphorylated region that engages the basic surface of RanGTP. CBC engagement in this complex is incompatible with ALYREF or NCBP3 interactions, displaces ARS2 from CBC, and synergistic binding of all components is required for export complex formation.","method":"Cryo-EM structure determination, in vitro mutagenesis, in-cell mutagenesis experiments","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with in vitro and in-cell mutagenesis validation, multiple orthogonal methods in a single study","pmids":["bio_10.1101_2024.11.28.625805"],"is_preprint":true},{"year":2024,"finding":"PHAX directly binds LIN28B and enhances LIN28B-mediated stabilization of PBX3 mRNA in esophageal cancer cells; PHAX knockdown reduces PBX3 mRNA stability, inhibits cancer cell proliferation, and promotes apoptosis and autophagy in vitro and in vivo.","method":"Co-immunoprecipitation (PHAX–LIN28B interaction), RNA immunoprecipitation, siRNA knockdown with proliferation/apoptosis/autophagy readouts, mouse xenograft","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP plus in vitro and in vivo functional assays, single lab, mechanistic claim supported by multiple readouts but in a cancer-specific context","pmids":["39668567"],"is_preprint":false},{"year":2024,"finding":"In glioma, HRasV12 activates PHAX, which recruits TRIM24 and Ku-dependent DNA-PKcs to U3 snoRNAs; DNA-PKcs then phosphorylates TRIM24 at S767/768, driving epigenome reprogramming. This places PHAX as a scaffold linking U3 snoRNA to a kinase signaling cascade in oncogenesis.","method":"Co-immunoprecipitation, RNA immunoprecipitation, phosphorylation site mutagenesis, single-cell RNA-seq, pharmacological inhibition (NU7441)","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP and RIP with phospho-mutagenesis and pharmacological rescue, single lab, complex multi-component mechanism","pmids":["38828688"],"is_preprint":false},{"year":2022,"finding":"In Drosophila, loss of Phax causes widespread alternative splicing changes; genetic suppressor analysis shows that allele-specific alternative splicing (intron retention vs. splicing) of Ribosomal protein S21 (RpS21) can fully suppress larval lethality of Phax mutants, placing RpS21 splicing downstream of Phax function in snRNP biogenesis.","method":"Genetic epistasis/suppressor analysis in Drosophila, RT-PCR for splicing, transposon mutant characterization","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with clean suppressor phenotype in Drosophila ortholog, single lab","pmids":["35920767"],"is_preprint":false}],"current_model":"PHAX is a phosphorylated adaptor protein that assembles onto m7G-capped short Pol II transcripts (U snRNAs, certain snoRNA precursors, short mRNAs) via its novel helical RNA-binding domain and interactions with CBC; phosphorylation is required for export complex assembly with CRM1–RanGTP (as defined by a cryo-EM structure), while cytoplasmic dephosphorylation drives disassembly and recycling; DDX39B/UAP56 loads PHAX onto RNA in an ATP-dependent manner via ALYREF; hnRNP C tetramers exclude PHAX from long transcripts to enforce length-dependent RNA class identity; and beyond snRNA export, PHAX promotes intranuclear routing of snoRNAs to Cajal bodies, facilitates nuclear export of short mRNAs such as H2AX (supporting the DNA damage response), and can scaffold additional signaling complexes on snoRNAs in certain oncogenic contexts."},"narrative":{"mechanistic_narrative":"PHAX (phosphorylated adaptor for RNA export) is the central scaffold that licenses m7G-capped short Pol II transcripts—principally U snRNAs—for CRM1-dependent nuclear export, coupling cap recognition to the export machinery in a phosphorylation-gated manner [PMID:10786834]. It is built around a novel, evolutionarily conserved helical RNA-binding domain that folds only upon engaging single-stranded RNA without sequence specificity, plus a distinct CBC-interaction region and nuclear localization signals that together let PHAX bridge CBC-bound capped RNA to the export receptor [PMID:11333016, PMID:20430857]. Phosphorylation is required for export complex assembly while cytoplasmic dephosphorylation drives disassembly, imposing directionality on transport [PMID:10786834]; cryo-EM of the assembled snRNA export complex shows the central region of PHAX bridging the capped RNA–CBC to CRM1–RanGTP and contacting CRM1 through a phosphorylated segment, with CBC engagement displacing ARS2 and excluding ALYREF/NCBP3 [PMID:bio_10.1101_2024.11.28.625805]. PHAX is actively loaded onto U snRNA by the ATP-dependent helicase UAP56/DDX39B via an ALYREF bridge, a route distinct from bulk mRNA export [PMID:39011894], and is selectively excluded from longer transcripts by hnRNP C tetramers on CBC, enforcing length-dependent RNA class identity [PMID:36620872]. Beyond export, PHAX directs intranuclear routing of m7G-capped box C/D snoRNA precursors to Cajal bodies as the first step of their maturation [PMID:15574332], and supports the DNA damage response by promoting transcription and nuclear export of the short H2AX mRNA [PMID:32759388]. In specific oncogenic contexts PHAX additionally scaffolds signaling complexes—linking U3 snoRNA to a TRIM24/DNA-PKcs cascade in glioma and binding LIN28B to stabilize PBX3 mRNA in esophageal cancer [PMID:38828688, PMID:39668567].","teleology":[{"year":2000,"claim":"Established PHAX as an essential, phosphorylation-controlled adaptor that physically couples capped U snRNA to the CRM1 export pathway and explains export directionality.","evidence":"In vitro export complex assembly, in vivo depletion, and biochemical analysis of phosphorylation state","pmids":["10786834"],"confidence":"High","gaps":["Kinase and phosphatase that toggle PHAX phosphorylation not identified","Structural basis of CBC/CRM1 bridging not resolved at this stage"]},{"year":2001,"claim":"Defined the modular architecture—a conserved RNA-binding domain, separable CBC-interaction region, and NLSs—that allows PHAX to act as a recyclable export scaffold.","evidence":"Systematic domain mutagenesis with in vivo export and binding assays","pmids":["11333016"],"confidence":"High","gaps":["Atomic structure of the RBD not yet determined","How NLS-driven recycling is coordinated with dephosphorylation unclear"]},{"year":2004,"claim":"Extended PHAX function beyond export to intranuclear trafficking, showing it routes capped box C/D snoRNA precursors to Cajal bodies, with cap hypermethylation blocking their export.","evidence":"RNA–protein co-IP, in vivo inactivation of PHAX and CRM1, fluorescence microscopy","pmids":["15574332"],"confidence":"High","gaps":["Molecular signal distinguishing Cajal-body routing from export not defined","Cap hypermethylation enzymology not addressed here"]},{"year":2010,"claim":"Solved the RBD fold, revealing a novel helical domain that is intrinsically disordered until RNA binding induces tertiary structure, and confirmed RNA binding is required for export.","evidence":"NMR, X-ray crystallography, and mutational analysis with export assays","pmids":["20430857"],"confidence":"High","gaps":["Lack of sequence specificity leaves substrate selection to other factors","Structure of the RBD within the full export complex not yet known"]},{"year":2020,"claim":"Connected PHAX to genome stability by showing it controls both transcription and export of the short H2AX mRNA, with loss sensitizing cells to DNA damage.","evidence":"siRNA knockdown, RT-qPCR/Northern blot, γH2AX immunofluorescence, damage sensitivity assays","pmids":["32759388"],"confidence":"Medium","gaps":["Mechanism by which PHAX affects H2AX transcription unresolved","Generality across other short mRNAs not established"]},{"year":2022,"claim":"Placed PHAX function genetically upstream of snRNP-dependent splicing in vivo, showing a single splicing event (RpS21 intron retention) can rescue Phax-null lethality.","evidence":"Drosophila genetic suppressor/epistasis analysis with RT-PCR","pmids":["35920767"],"confidence":"Medium","gaps":["Direct molecular link from PHAX to RpS21 splicing not shown","Relevance to mammalian PHAX splicing roles untested"]},{"year":2023,"claim":"Defined how RNA class identity is enforced upstream of PHAX: hnRNP C tetramers on CBC-bound long transcripts block PHAX recruitment, partitioning them to the mRNA pathway.","evidence":"Co-IP of CBC–hnRNP C, hnRNP C tetramerization/RNA-binding mutants, in vivo export assays","pmids":["36620872"],"confidence":"Medium","gaps":["Precise length threshold and how it is read out not defined","Single-lab data without reciprocal structural validation"]},{"year":2024,"claim":"Identified the ATP-dependent loading mechanism that places PHAX on U snRNA, with UAP56/DDX39B stimulating RNA binding and ALYREF bridging the two, defining a TREX-linked route distinct from mRNA export.","evidence":"In vitro ATP-dependent factor assay, RNA-binding and co-IP, in vivo export assays","pmids":["39011894"],"confidence":"High","gaps":["How ALYREF is later displaced during CRM1 complex assembly not shown here","Stoichiometry of the loading intermediate unresolved"]},{"year":2024,"claim":"Provided the structural mechanism of the complete export complex, showing phosphorylated PHAX bridges capped RNA–CBC to CRM1–RanGTP, reinforces cap binding, and remodels CBC by displacing ARS2 and excluding ALYREF/NCBP3.","evidence":"Cryo-EM structure with in vitro and in-cell mutagenesis (preprint)","pmids":["bio_10.1101_2024.11.28.625805"],"confidence":"High","gaps":["Transition from the ALYREF-bound loading intermediate to this complex not visualized","Dephosphorylation-driven disassembly state not structurally captured"]},{"year":2024,"claim":"Revealed non-canonical scaffolding roles for PHAX in oncogenesis, linking it to a U3 snoRNA–TRIM24–DNA-PKcs signaling cascade in glioma and to LIN28B-mediated PBX3 mRNA stabilization in esophageal cancer.","evidence":"Co-IP, RIP, phospho-site mutagenesis, single-cell RNA-seq, pharmacological inhibition, knockdown with proliferation/apoptosis assays and xenograft","pmids":["38828688","39668567"],"confidence":"Medium","gaps":["Whether these activities depend on canonical PHAX export function is unclear","Context-specificity and generality across tumor types not established"]},{"year":null,"claim":"How the kinase/phosphatase cycle, helicase-driven loading, and CBC remodeling are temporally coordinated into a single directional export cycle remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["Identity of the PHAX kinase and cytoplasmic phosphatase unknown","No structure of the loading intermediate or disassembly state","Mechanistic basis of PHAX's effects on transcription not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,1,3,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,7]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,2]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,2,6]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,7]}],"complexes":["U snRNA export complex (PHAX–CBC–CRM1–RanGTP)"],"partners":["NCBP1","NCBP2","XPO1","RAN","DDX39B","ALYREF","HNRNPC","LIN28B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H814","full_name":"Phosphorylated adapter RNA export protein","aliases":["RNA U small nuclear RNA export adapter protein"],"length_aa":394,"mass_kda":44.4,"function":"A phosphoprotein adapter involved in the XPO1-mediated U snRNA export from the nucleus (PubMed:39011894). Bridge components required for U snRNA export, the cap binding complex (CBC)-bound snRNA on the one hand and the GTPase Ran in its active GTP-bound form together with the export receptor XPO1 on the other. Its phosphorylation in the nucleus is required for U snRNA export complex assembly and export, while its dephosphorylation in the cytoplasm causes export complex disassembly. It is recycled back to the nucleus via the importin alpha/beta heterodimeric import receptor. The directionality of nuclear export is thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. Its compartmentalized phosphorylation cycle may also contribute to the directionality of export. Binds strongly to m7G-capped U1 and U5 small nuclear RNAs (snRNAs) in a sequence-unspecific manner and phosphorylation-independent manner (By similarity). Also plays a role in the biogenesis of U3 small nucleolar RNA (snoRNA). Involved in the U3 snoRNA transport from nucleoplasm to Cajal bodies. Binds strongly to m7G-capped U3, U8 and U13 precursor snoRNAs and weakly to trimethylated (TMG)-capped U3, U8 and U13 snoRNAs. Also binds to telomerase RNA","subcellular_location":"Nucleus, nucleoplasm; Nucleus, Cajal body; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9H814/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PHAX","classification":"Common Essential","n_dependent_lines":1191,"n_total_lines":1208,"dependency_fraction":0.9859271523178808},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000164902","cell_line_id":"CID001569","localizations":[{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"SRRT","stoichiometry":10.0},{"gene":"YTHDF2","stoichiometry":10.0},{"gene":"NCBP1","stoichiometry":10.0},{"gene":"NFYA","stoichiometry":4.0},{"gene":"LIMD1","stoichiometry":4.0},{"gene":"DDX39B","stoichiometry":0.2},{"gene":"KPNA1","stoichiometry":0.2},{"gene":"KPNA2","stoichiometry":0.2},{"gene":"KPNA3","stoichiometry":0.2},{"gene":"KPNA4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001569","total_profiled":1310},"omim":[{"mim_id":"621061","title":"LEUKODYSTROPHY, DEMYELINATING, ADULT-ONSET, AUTOSOMAL DOMINANT, ATYPICAL; ADLDAT","url":"https://www.omim.org/entry/621061"},{"mim_id":"604924","title":"PHOSPHORYLATED ADAPTOR FOR RNA EXPORT; PHAX","url":"https://www.omim.org/entry/604924"},{"mim_id":"602394","title":"NUCLEOLAR AND COILED-BODY PHOSPHOPROTEIN 1; NOLC1","url":"https://www.omim.org/entry/602394"},{"mim_id":"600354","title":"SURVIVAL OF MOTOR NEURON 1; SMN1","url":"https://www.omim.org/entry/600354"},{"mim_id":"169500","title":"LEUKODYSTROPHY, DEMYELINATING, ADULT-ONSET, AUTOSOMAL DOMINANT, TYPICAL; ADLDTY","url":"https://www.omim.org/entry/169500"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PHAX"},"hgnc":{"alias_symbol":["FLJ13193"],"prev_symbol":["RNUXA"]},"alphafold":{"accession":"Q9H814","domains":[{"cath_id":"1.10.10.1440","chopping":"208-299","consensus_level":"high","plddt":89.0664,"start":208,"end":299},{"cath_id":"1.20.5","chopping":"301-340","consensus_level":"medium","plddt":85.4103,"start":301,"end":340}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H814","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H814-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H814-F1-predicted_aligned_error_v6.png","plddt_mean":65.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PHAX","jax_strain_url":"https://www.jax.org/strain/search?query=PHAX"},"sequence":{"accession":"Q9H814","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H814.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H814/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H814"}},"corpus_meta":[{"pmid":"10786834","id":"PMC_10786834","title":"PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation.","date":"2000","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/10786834","citation_count":291,"is_preprint":false},{"pmid":"15574332","id":"PMC_15574332","title":"PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli.","date":"2004","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/15574332","citation_count":144,"is_preprint":false},{"pmid":"11333016","id":"PMC_11333016","title":"The evolutionarily conserved region of the U snRNA export mediator PHAX is a novel RNA-binding domain that is essential for U snRNA export.","date":"2001","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/11333016","citation_count":37,"is_preprint":false},{"pmid":"25195018","id":"PMC_25195018","title":"A syndromic form of Pierre Robin sequence is caused by 5q23 deletions encompassing FBN2 and PHAX.","date":"2014","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25195018","citation_count":20,"is_preprint":false},{"pmid":"20430857","id":"PMC_20430857","title":"Structure and RNA recognition by the snRNA and snoRNA transport factor PHAX.","date":"2010","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/20430857","citation_count":14,"is_preprint":false},{"pmid":"36620872","id":"PMC_36620872","title":"The hnRNP C tetramer binds to CBC on mRNA and impedes PHAX recruitment for the classification of RNA polymerase II transcripts.","date":"2023","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/36620872","citation_count":12,"is_preprint":false},{"pmid":"38828688","id":"PMC_38828688","title":"TRIM24 Cooperates with Ras Mutation to Drive Glioma Progression through snoRNA Recruitment of PHAX and DNA-PKcs.","date":"2024","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/38828688","citation_count":10,"is_preprint":false},{"pmid":"32759388","id":"PMC_32759388","title":"The RNA transport factor PHAX is required for proper histone H2AX expression and DNA damage response.","date":"2020","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/32759388","citation_count":8,"is_preprint":false},{"pmid":"39011894","id":"PMC_39011894","title":"The RNA helicase DDX39 contributes to the nuclear export of spliceosomal U snRNA by loading of PHAX onto RNA.","date":"2024","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/39011894","citation_count":7,"is_preprint":false},{"pmid":"30759165","id":"PMC_30759165","title":"Elevated levels of autoantibodies against EXD2 and PHAX in the sera of patients with chronic thromboembolic pulmonary hypertension.","date":"2019","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30759165","citation_count":5,"is_preprint":false},{"pmid":"35920767","id":"PMC_35920767","title":"Allele-specific alternative splicing of Drosophila Ribosomal protein S21 suppresses a lethal mutation in the Phosphorylated adaptor for RNA export (Phax) gene.","date":"2022","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/35920767","citation_count":4,"is_preprint":false},{"pmid":"39668567","id":"PMC_39668567","title":"PHAX enhanced LIN28B-mediated PBX3 mRNA stability to promote esophageal cancer development.","date":"2024","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/39668567","citation_count":4,"is_preprint":false},{"pmid":"32272660","id":"PMC_32272660","title":"The Efficacy of Sunitinib Treatment of Renal Cancer Cells Is Associated with the Protein PHAX In Vitro.","date":"2020","source":"Biology","url":"https://pubmed.ncbi.nlm.nih.gov/32272660","citation_count":3,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.11.28.625805","title":"Structural basis for the synergistic assembly of the snRNA export complex","date":"2024-11-28","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.28.625805","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8141,"output_tokens":3001,"usd":0.034719,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10446,"output_tokens":3486,"usd":0.06969,"stage2_stop_reason":"end_turn"},"total_usd":0.104409,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"PHAX (phosphorylated adaptor for RNA export) is an essential factor for U snRNA nuclear export complex assembly in vitro; it directly contacts RNA, the nuclear cap-binding complex (CBC), and the export receptor CRM1/Xpo1, and requires phosphorylation for export complex assembly. Dephosphorylation in the cytoplasm causes export complex disassembly, providing directionality.\",\n      \"method\": \"In vitro export complex assembly assay, in vivo knockdown/depletion, identification of phosphorylation state by biochemistry\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstituted in vitro export complex, in vivo depletion with specific phenotype, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"10786834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The most evolutionarily conserved region of PHAX is a novel RNA-binding domain (RBD) essential for U snRNA export. PHAX also contains two major nuclear localization signals (NLSs) required for nuclear recycling after export, and a CBC interaction domain at least partly distinct from the RBD and NLSs, allowing PHAX to act as a scaffold for U snRNA export complex assembly.\",\n      \"method\": \"Systematic mutagenesis/deletion analysis of PHAX domains combined with in vivo export assays and binding assays\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — domain mutagenesis with functional in vivo and in vitro validation, multiple orthogonal methods\",\n      \"pmids\": [\"11333016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PHAX binds m7G-capped U3 snoRNA precursors (as well as U8, U13 box C/D snoRNA precursors and telomerase RNA) and is required for transport of U3 snoRNA to Cajal bodies in the first step of intranuclear routing; CRM1 is subsequently required for routing from Cajal bodies to nucleoli. PHAX does not export m7G-capped U3 because its cap becomes hypermethylated in the nucleus.\",\n      \"method\": \"Immunoprecipitation of RNA–protein complexes, inactivation of PHAX and CRM1 in vivo, fluorescence microscopy\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP/RIP combined with in vivo inactivation and microscopy, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15574332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The PHAX RNA-binding domain (RNA_GG_bind domain) adopts a novel helical fold and is monomeric in the absence of ligand, only adopting a tertiary structure upon RNA binding; it binds single-stranded RNA with micromolar affinity without sequence specificity. Mutational analysis confirmed that RNA-binding by PHAX-RBD is required for PHAX-mediated nuclear export.\",\n      \"method\": \"NMR spectroscopy, X-ray crystallography, mutational analysis of RNA binding\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR and crystal structure combined with mutagenesis and functional export assay, single lab\",\n      \"pmids\": [\"20430857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PHAX is required for efficient DNA damage response (DDR): 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 cellular sensitivity to DNA damage.\",\n      \"method\": \"siRNA knockdown of PHAX, RT-qPCR/Northern blot for H2AX mRNA, γH2AX immunofluorescence, DNA damage sensitivity assays\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotype and mRNA-level mechanistic readout, single lab, multiple readouts\",\n      \"pmids\": [\"32759388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The CBC directly interacts with hnRNP C on mRNA, and the tetramer-forming activity and strong RNA-binding activity of hnRNP C together impede PHAX recruitment to longer transcripts, thereby directing them to the mRNA export pathway rather than the U snRNA export pathway. This reveals the molecular mechanism by which length-dependent RNA classification excludes PHAX from long Pol II transcripts.\",\n      \"method\": \"Co-immunoprecipitation of CBC–hnRNP C complex on mRNA, RNA-binding and tetramerization mutants of hnRNP C, in vivo export assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with mutational dissection, single lab, multiple orthogonal assays\",\n      \"pmids\": [\"36620872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The RNA helicase UAP56/DDX39B (and its paralog URH49/DDX39A) stimulates RNA binding of PHAX in an ATP-dependent manner, thereby loading PHAX onto U snRNA and promoting U snRNA export. ALYREF acts as a bridge between PHAX and UAP56/DDX39B. This TREX-component-dependent mechanism for U snRNA export is distinct from the mRNA export mechanism.\",\n      \"method\": \"In vitro ATP-dependent factor identification assay, RNA-binding assays, co-immunoprecipitation, in vivo U snRNA export assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — novel in vitro reconstitution strategy plus co-IP and in vivo functional validation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39011894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of the complete snRNA export complex (phosphorylated PHAX + CBC + CRM1 + Ran-GTP + capped RNA) reveals that the central region of PHAX bridges CBC-bound capped RNA to CRM1–RanGTP, reinforces cap dinucleotide binding, and contacts CRM1 via a phosphorylated region that engages the basic surface of RanGTP. CBC engagement in this complex is incompatible with ALYREF or NCBP3 interactions, displaces ARS2 from CBC, and synergistic binding of all components is required for export complex formation.\",\n      \"method\": \"Cryo-EM structure determination, in vitro mutagenesis, in-cell mutagenesis experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with in vitro and in-cell mutagenesis validation, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"bio_10.1101_2024.11.28.625805\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PHAX directly binds LIN28B and enhances LIN28B-mediated stabilization of PBX3 mRNA in esophageal cancer cells; PHAX knockdown reduces PBX3 mRNA stability, inhibits cancer cell proliferation, and promotes apoptosis and autophagy in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation (PHAX–LIN28B interaction), RNA immunoprecipitation, siRNA knockdown with proliferation/apoptosis/autophagy readouts, mouse xenograft\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP plus in vitro and in vivo functional assays, single lab, mechanistic claim supported by multiple readouts but in a cancer-specific context\",\n      \"pmids\": [\"39668567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In glioma, HRasV12 activates PHAX, which recruits TRIM24 and Ku-dependent DNA-PKcs to U3 snoRNAs; DNA-PKcs then phosphorylates TRIM24 at S767/768, driving epigenome reprogramming. This places PHAX as a scaffold linking U3 snoRNA to a kinase signaling cascade in oncogenesis.\",\n      \"method\": \"Co-immunoprecipitation, RNA immunoprecipitation, phosphorylation site mutagenesis, single-cell RNA-seq, pharmacological inhibition (NU7441)\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP and RIP with phospho-mutagenesis and pharmacological rescue, single lab, complex multi-component mechanism\",\n      \"pmids\": [\"38828688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Drosophila, loss of Phax causes widespread alternative splicing changes; genetic suppressor analysis shows that allele-specific alternative splicing (intron retention vs. splicing) of Ribosomal protein S21 (RpS21) can fully suppress larval lethality of Phax mutants, placing RpS21 splicing downstream of Phax function in snRNP biogenesis.\",\n      \"method\": \"Genetic epistasis/suppressor analysis in Drosophila, RT-PCR for splicing, transposon mutant characterization\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with clean suppressor phenotype in Drosophila ortholog, single lab\",\n      \"pmids\": [\"35920767\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PHAX is a phosphorylated adaptor protein that assembles onto m7G-capped short Pol II transcripts (U snRNAs, certain snoRNA precursors, short mRNAs) via its novel helical RNA-binding domain and interactions with CBC; phosphorylation is required for export complex assembly with CRM1–RanGTP (as defined by a cryo-EM structure), while cytoplasmic dephosphorylation drives disassembly and recycling; DDX39B/UAP56 loads PHAX onto RNA in an ATP-dependent manner via ALYREF; hnRNP C tetramers exclude PHAX from long transcripts to enforce length-dependent RNA class identity; and beyond snRNA export, PHAX promotes intranuclear routing of snoRNAs to Cajal bodies, facilitates nuclear export of short mRNAs such as H2AX (supporting the DNA damage response), and can scaffold additional signaling complexes on snoRNAs in certain oncogenic contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PHAX (phosphorylated adaptor for RNA export) is the central scaffold that licenses m7G-capped short Pol II transcripts—principally U snRNAs—for CRM1-dependent nuclear export, coupling cap recognition to the export machinery in a phosphorylation-gated manner [#0]. It is built around a novel, evolutionarily conserved helical RNA-binding domain that folds only upon engaging single-stranded RNA without sequence specificity, plus a distinct CBC-interaction region and nuclear localization signals that together let PHAX bridge CBC-bound capped RNA to the export receptor [#1, #3]. Phosphorylation is required for export complex assembly while cytoplasmic dephosphorylation drives disassembly, imposing directionality on transport [#0]; cryo-EM of the assembled snRNA export complex shows the central region of PHAX bridging the capped RNA–CBC to CRM1–RanGTP and contacting CRM1 through a phosphorylated segment, with CBC engagement displacing ARS2 and excluding ALYREF/NCBP3 [#7]. PHAX is actively loaded onto U snRNA by the ATP-dependent helicase UAP56/DDX39B via an ALYREF bridge, a route distinct from bulk mRNA export [#6], and is selectively excluded from longer transcripts by hnRNP C tetramers on CBC, enforcing length-dependent RNA class identity [#5]. Beyond export, PHAX directs intranuclear routing of m7G-capped box C/D snoRNA precursors to Cajal bodies as the first step of their maturation [#2], and supports the DNA damage response by promoting transcription and nuclear export of the short H2AX mRNA [#4]. In specific oncogenic contexts PHAX additionally scaffolds signaling complexes—linking U3 snoRNA to a TRIM24/DNA-PKcs cascade in glioma and binding LIN28B to stabilize PBX3 mRNA in esophageal cancer [#9, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established PHAX as an essential, phosphorylation-controlled adaptor that physically couples capped U snRNA to the CRM1 export pathway and explains export directionality.\",\n      \"evidence\": \"In vitro export complex assembly, in vivo depletion, and biochemical analysis of phosphorylation state\",\n      \"pmids\": [\"10786834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase and phosphatase that toggle PHAX phosphorylation not identified\", \"Structural basis of CBC/CRM1 bridging not resolved at this stage\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined the modular architecture—a conserved RNA-binding domain, separable CBC-interaction region, and NLSs—that allows PHAX to act as a recyclable export scaffold.\",\n      \"evidence\": \"Systematic domain mutagenesis with in vivo export and binding assays\",\n      \"pmids\": [\"11333016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the RBD not yet determined\", \"How NLS-driven recycling is coordinated with dephosphorylation unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Extended PHAX function beyond export to intranuclear trafficking, showing it routes capped box C/D snoRNA precursors to Cajal bodies, with cap hypermethylation blocking their export.\",\n      \"evidence\": \"RNA–protein co-IP, in vivo inactivation of PHAX and CRM1, fluorescence microscopy\",\n      \"pmids\": [\"15574332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular signal distinguishing Cajal-body routing from export not defined\", \"Cap hypermethylation enzymology not addressed here\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Solved the RBD fold, revealing a novel helical domain that is intrinsically disordered until RNA binding induces tertiary structure, and confirmed RNA binding is required for export.\",\n      \"evidence\": \"NMR, X-ray crystallography, and mutational analysis with export assays\",\n      \"pmids\": [\"20430857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lack of sequence specificity leaves substrate selection to other factors\", \"Structure of the RBD within the full export complex not yet known\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected PHAX to genome stability by showing it controls both transcription and export of the short H2AX mRNA, with loss sensitizing cells to DNA damage.\",\n      \"evidence\": \"siRNA knockdown, RT-qPCR/Northern blot, γH2AX immunofluorescence, damage sensitivity assays\",\n      \"pmids\": [\"32759388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which PHAX affects H2AX transcription unresolved\", \"Generality across other short mRNAs not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed PHAX function genetically upstream of snRNP-dependent splicing in vivo, showing a single splicing event (RpS21 intron retention) can rescue Phax-null lethality.\",\n      \"evidence\": \"Drosophila genetic suppressor/epistasis analysis with RT-PCR\",\n      \"pmids\": [\"35920767\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link from PHAX to RpS21 splicing not shown\", \"Relevance to mammalian PHAX splicing roles untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined how RNA class identity is enforced upstream of PHAX: hnRNP C tetramers on CBC-bound long transcripts block PHAX recruitment, partitioning them to the mRNA pathway.\",\n      \"evidence\": \"Co-IP of CBC–hnRNP C, hnRNP C tetramerization/RNA-binding mutants, in vivo export assays\",\n      \"pmids\": [\"36620872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Precise length threshold and how it is read out not defined\", \"Single-lab data without reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified the ATP-dependent loading mechanism that places PHAX on U snRNA, with UAP56/DDX39B stimulating RNA binding and ALYREF bridging the two, defining a TREX-linked route distinct from mRNA export.\",\n      \"evidence\": \"In vitro ATP-dependent factor assay, RNA-binding and co-IP, in vivo export assays\",\n      \"pmids\": [\"39011894\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ALYREF is later displaced during CRM1 complex assembly not shown here\", \"Stoichiometry of the loading intermediate unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the structural mechanism of the complete export complex, showing phosphorylated PHAX bridges capped RNA–CBC to CRM1–RanGTP, reinforces cap binding, and remodels CBC by displacing ARS2 and excluding ALYREF/NCBP3.\",\n      \"evidence\": \"Cryo-EM structure with in vitro and in-cell mutagenesis (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.11.28.625805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transition from the ALYREF-bound loading intermediate to this complex not visualized\", \"Dephosphorylation-driven disassembly state not structurally captured\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed non-canonical scaffolding roles for PHAX in oncogenesis, linking it to a U3 snoRNA–TRIM24–DNA-PKcs signaling cascade in glioma and to LIN28B-mediated PBX3 mRNA stabilization in esophageal cancer.\",\n      \"evidence\": \"Co-IP, RIP, phospho-site mutagenesis, single-cell RNA-seq, pharmacological inhibition, knockdown with proliferation/apoptosis assays and xenograft\",\n      \"pmids\": [\"38828688\", \"39668567\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether these activities depend on canonical PHAX export function is unclear\", \"Context-specificity and generality across tumor types not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the kinase/phosphatase cycle, helicase-driven loading, and CBC remodeling are temporally coordinated into a single directional export cycle remains unresolved.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the PHAX kinase and cytoplasmic phosphatase unknown\", \"No structure of the loading intermediate or disassembly state\", \"Mechanistic basis of PHAX's effects on transcription not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1, 3, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 2, 6]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"complexes\": [\"U snRNA export complex (PHAX–CBC–CRM1–RanGTP)\"],\n    \"partners\": [\"NCBP1\", \"NCBP2\", \"XPO1\", \"RAN\", \"DDX39B\", \"ALYREF\", \"HNRNPC\", \"LIN28B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}