{"gene":"RPF1","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2002,"finding":"RPF1 (yeast) belongs to the Imp4 superfamily of RNA-binding proteins that all possess the sigma(70)-like motif, a eukaryotic RNA binding domain with prokaryotic origins. Members of this superfamily associate with RNAs consistent with their distinct roles in ribosome biogenesis, and yeast Rpf1 is required for a specific stage of ribosome biogenesis.","method":"Bioinformatic identification of conserved sigma(70)-like RNA binding domain; functional requirement established by genetic analysis in yeast","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain identification combined with genetic requirement for ribosome biogenesis, single study","pmids":["11864606"],"is_preprint":false},{"year":2006,"finding":"Kap120 functions as a nuclear import receptor for yeast Rpf1 (ribosome assembly factor). Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1 (via Kap120, Kap114, and Nmd5). Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus; overexpression of RPF1 suppresses the nuclear 60S accumulation in kap120 mutants, indicating Kap120 functions in nuclear import of Rpf1 rather than in ribosomal export directly.","method":"Two-hybrid screen, in vitro direct binding assay, Ran-GTP release assay, genetic epistasis (deletion mutants, overexpression suppression), nuclear accumulation phenotype assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal in vitro binding, Ran-GTP dissociation, genetic epistasis with overexpression suppression, multiple orthogonal methods in one study","pmids":["16581791"],"is_preprint":false},{"year":2013,"finding":"Yeast Rpf1 is genetically linked to the DEAD-box RNA helicase Mak5, ribosomal protein Rpl14, and 60S biogenesis factors Ebp2 and Nop16 (synthetic lethality). Mak5 is associated with the Nsa1 pre-60S particle. These 'Mak5 cluster' factors, including Rpf1, are proposed to orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface (Rpl6, Rpl14, Rpl16 and rRNA expansion segments ES7L and ES39L).","method":"Synthetic lethal genetic screens with mak5 alleles; co-immunoprecipitation of Mak5 with the Nsa1 pre-60S particle","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — synthetic lethality screen and co-IP placing Rpf1 in a defined genetic/physical network, single lab","pmids":["24312670"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structures of early nucleolar pre-60S biogenesis intermediates at 3.3–4.5 Å resolution reveal that yeast Rpf1 is part of the Nsa1-Rrp1-Rpf1-Mak16 module, which stabilizes the solvent side of the 60S subunit during early nucleolar assembly. Rpf1 is mapped within six different assembly states, showing its position and integration order in the sequential folding pathway.","method":"Cryo-EM structural analysis of six pre-60S assembly intermediates; mapping of 25 assembly factors","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures at near-atomic resolution with multiple assembly states, direct mapping of Rpf1 in defined module, published in high-profile journal","pmids":["29245012"],"is_preprint":false},{"year":2024,"finding":"Human RPF1 is required for accurate 60S ribosome biogenesis. siRNA-mediated knockdown of RPF1 in HEK293 cells significantly changed the pattern of RNA products derived from 47S pre-rRNA. RPF1 is associated with pre-60S particles (not pre-40S). RPF1 is not directly involved in pre-rRNA cleavage but rather helps pre-rRNA acquire the conformation favoring cleavage.","method":"shRNA/siRNA knockdown in HEK293 cells; pre-rRNA processing pattern analysis; pre-ribosomal particle association assay","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined pre-rRNA processing phenotype and particle association, single lab, single study","pmids":["38391939"],"is_preprint":false},{"year":2024,"finding":"Human RPF1 functions as part of a pre-ribosomal WDR74 module (consisting of WDR74, RPF1, MAK16, and RRP1) that is mutually required for interactions with MTR4. Each component of the WDR74 module is mutually essential for the interaction of other members with MTR4, and all components are required for accurate cleavage of pre-rRNA during 60S ribosome biogenesis.","method":"Co-immunoprecipitation combined with mass spectrometry; functional knockdown analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP/MS identifying the complex and functional requirement established, single lab","pmids":["39706051"],"is_preprint":false},{"year":2025,"finding":"Yeast Mak16 contains a [4Fe-4S] cluster and its interaction with Rpf1 is functionally dependent on Fe/S cluster integrity. Oxidative stress (H2O2) destabilizes Mak16 and disrupts its interaction with Rpf1 in vivo. Disruption of Fe/S cluster coordination in Mak16 impairs the Mak16-Rpf1 complex formation and decreases 25S rRNA level. Thus Rpf1 participates in a redox-sensitive step of early 60S maturation via its partner Mak16.","method":"In vivo and in vitro Fe/S cluster characterization; co-immunoprecipitation to detect Mak16-Rpf1 interaction under oxidative stress; rRNA level measurement after Fe/S cluster disruption mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (in vitro biochemistry, in vivo co-IP, mutagenesis, rRNA quantification) in a single rigorous study establishing the functional Mak16-Rpf1 interaction","pmids":["41231949"],"is_preprint":false},{"year":1996,"finding":"RPF-1 (POU6F2) encodes a POU-domain transcription factor expressed in retinal ganglion and amacrine cells. In vitro, the RPF-1 POU domain (without the alternatively spliced insert) binds to a consensus Oct-1 binding site, whereas the alternatively spliced isoform (with a 36 amino acid insert in the POU-specific domain DNA recognition helix) does not bind this site. RPF-1 protein first appears at e11 in mouse retina, localizing to neuroblasts that have recently migrated to the future ganglion cell layer.","method":"In vitro DNA binding assay (POU domain binding to Oct-1 consensus); cDNA/genomic cloning; in situ hybridization for subcellular/tissue localization","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — direct in vitro binding assay with mutagenesis-like isoform comparison, combined with developmental localization, single lab","pmids":["8601806"],"is_preprint":false},{"year":2016,"finding":"Human RPF-1 (POU6F2) shows nuclear localization in HEK293 stable transfectants, elevated protein stability, and transactivation of promoters featuring POU consensus sites. RPF-1 induction leads to reduced cell proliferation and reduced in vivo tumor growth. Gel-shift competition assay demonstrated specificity of RPF-1 binding to consensus POU motifs, and showed that the Ser-rich region upstream of the POU domain is indispensable for DNA binding. Genome-wide ChIP-chip analysis predicted promoter occupancy consistent with transcriptome changes, identifying 217 candidate target genes.","method":"Stable inducible transfection; nuclear localization by direct imaging; transactivation reporter assay; gel-shift/EMSA with competition; deletion mutagenesis of Ser-rich region; ChIP-chip; transcriptome profiling; tumor xenograft growth assay","journal":"The international journal of biochemistry & cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (EMSA with mutagenesis, reporter assay, ChIP-chip, cellular and in vivo phenotype) in a single study","pmids":["27425396"],"is_preprint":false},{"year":2014,"finding":"Rat/porcine RPF1 (POU6F2) protein binds specifically to the 5'-upstream region of porcine Fshβ gene, identified by Yeast One-Hybrid System. RPF1 shows regulatory (transactivation) activity for Prop1 and Prrx2 promoters but not for Prrx1 in reporter assays. RPF1 mRNA is expressed in the stem/progenitor cells of the rat pituitary primordium and decreases during pituitary development.","method":"Yeast one-hybrid system; luciferase reporter assay; in situ hybridization; RT-PCR","journal":"The Journal of reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct DNA binding by yeast one-hybrid plus reporter assay for transcriptional activity, single lab","pmids":["24804940"],"is_preprint":false}],"current_model":"RPF1 encodes two functionally distinct proteins depending on context: in ribosome biogenesis (yeast Rpf1/human RPF1), it is a nucleolar assembly factor containing a sigma(70)-like RNA-binding domain that is imported into the nucleus via Kap120 (and parallel importins), associates with early pre-60S particles as part of the Nsa1-Rrp1-Rpf1-Mak16/WDR74 module to stabilize the solvent face of the 60S subunit, and is required for correct pre-rRNA processing and 25S rRNA accumulation; its partner Mak16 carries a redox-sensitive [4Fe-4S] cluster whose integrity is required for the Mak16-Rpf1 interaction. Separately, the human POU6F2/RPF-1 transcription factor uses its POU domain to bind octamer DNA consensus sites (with splice-isoform-dependent binding), transactivates target promoters including PROP1 and PRRX2 in developing pituitary and retina, requires its N-terminal Ser-rich region for DNA binding, and suppresses cell proliferation and tumor growth when induced."},"narrative":{"mechanistic_narrative":"The RPF1 symbol maps to two unrelated proteins in this corpus, and the timeline coheres into two internally consistent mechanistic pictures. In ribosome biogenesis, yeast and human RPF1 is a nucleolar pre-60S assembly factor: yeast Rpf1 belongs to the Imp4 superfamily defined by a sigma(70)-like RNA-binding domain and is required for a specific stage of large-subunit biogenesis [PMID:11864606]. It is delivered to the nucleus by the import receptor Kap120 (with parallel routes via Kap114 and Nmd5), as Kap120 binds Rpf1 directly and is released by Ran-GTP, and loss of either factor traps 60S particles in the nucleus [PMID:16581791]. Within early nucleolar pre-60S intermediates, Rpf1 is an integral member of the Nsa1-Rrp1-Rpf1-Mak16 module that stabilizes the solvent-exposed face of the maturing 60S subunit [PMID:29245012], a function that depends on the [4Fe-4S] cluster of its partner Mak16, whose oxidative disruption breaks the Mak16-Rpf1 interaction and lowers 25S rRNA levels, placing Rpf1 at a redox-sensitive step of 60S maturation [PMID:41231949]. Human RPF1 is the conserved counterpart, associating with pre-60S (not pre-40S) particles and acting within a WDR74-RPF1-MAK16-RRP1 module whose members are mutually required for their interaction with the helicase MTR4 and for accurate pre-rRNA cleavage; RPF1 itself does not catalyze cleavage but promotes the pre-rRNA conformation that favors it [PMID:38391939, PMID:39706051]. Distinctly, the POU6F2/RPF-1 entries describe a POU-domain transcription factor: it binds octamer/POU consensus DNA in an isoform-dependent manner requiring an N-terminal Ser-rich region [PMID:8601806, PMID:27425396], transactivates developmental promoters including Prop1 and Prrx2 in pituitary progenitors [PMID:24804940], and on induction localizes to the nucleus, suppresses proliferation, and reduces tumor xenograft growth [PMID:27425396].","teleology":[{"year":2002,"claim":"Established that yeast Rpf1 is an RNA-binding protein of the Imp4/sigma(70)-like superfamily functionally required for a defined stage of ribosome biogenesis, defining its molecular class.","evidence":"Bioinformatic domain identification plus genetic requirement analysis in yeast","pmids":["11864606"],"confidence":"Medium","gaps":["Did not define which biogenesis substep or partner particle","RNA target of the sigma(70)-like domain not directly mapped"]},{"year":2006,"claim":"Answered how Rpf1 reaches the nucleolus, showing Kap120 is a direct import receptor (with parallel Kap114/Nmd5 routes) and that the nuclear 60S accumulation defect reflects failed Rpf1 import rather than a direct export role.","evidence":"Two-hybrid, in vitro binding, Ran-GTP release, and genetic epistasis/overexpression suppression in yeast","pmids":["16581791"],"confidence":"High","gaps":["Import signal on Rpf1 not delimited","Relative contribution of the three parallel importins unquantified"]},{"year":2013,"claim":"Placed Rpf1 in a functional network with Mak5, Rpl14, Ebp2 and Nop16 implicated in shaping a eukaryote-specific 60S surface, linking it physically/genetically to the Nsa1 pre-60S particle.","evidence":"Synthetic lethal genetic screens and co-IP of Mak5 with the Nsa1 particle","pmids":["24312670"],"confidence":"Medium","gaps":["Direct Rpf1 contacts within the particle not resolved","Genetic interactions do not prove direct binding"]},{"year":2017,"claim":"Resolved Rpf1's structural position by mapping it within the Nsa1-Rrp1-Rpf1-Mak16 module that stabilizes the solvent face of the 60S subunit across sequential early nucleolar intermediates.","evidence":"Cryo-EM of six pre-60S assembly states at 3.3-4.5 Å with assembly-factor mapping","pmids":["29245012"],"confidence":"High","gaps":["Functional consequence of each contact not tested by mutagenesis","Order of module release during maturation not fully defined"]},{"year":2024,"claim":"Extended the role to human RPF1, showing it associates specifically with pre-60S particles and shapes pre-rRNA conformation to enable correct cleavage of 47S-derived precursors rather than catalyzing cleavage itself.","evidence":"siRNA/shRNA knockdown in HEK293, pre-rRNA processing analysis, particle association assays","pmids":["38391939"],"confidence":"Medium","gaps":["Direct RNA-binding by human RPF1 not demonstrated","Single cell line, single lab"]},{"year":2024,"claim":"Defined the human module as WDR74-RPF1-MAK16-RRP1 whose members are mutually required for engaging the helicase MTR4 and for accurate pre-rRNA cleavage, establishing interdependence within the module.","evidence":"Co-IP/mass spectrometry and functional knockdown","pmids":["39706051"],"confidence":"Medium","gaps":["Direct vs. indirect RPF1-MTR4 contact not distinguished","Stoichiometry and assembly order of the module unresolved"]},{"year":2025,"claim":"Identified a redox control point in early 60S maturation by showing the Mak16 [4Fe-4S] cluster is required for the Mak16-Rpf1 interaction, with oxidative stress disrupting the complex and lowering 25S rRNA.","evidence":"In vitro/in vivo Fe/S characterization, co-IP under H2O2, and rRNA quantification after cluster-disrupting mutagenesis in yeast","pmids":["41231949"],"confidence":"High","gaps":["Whether Rpf1 itself senses redox is not addressed","Physiological oxidative conditions that engage this switch not defined"]},{"year":1996,"claim":"Characterized the unrelated POU6F2/RPF-1 as a POU-domain transcription factor with isoform-dependent octamer DNA binding, expressed during retinal ganglion/amacrine cell development.","evidence":"In vitro DNA-binding assay with isoform comparison, cloning, and in situ hybridization in mouse retina","pmids":["8601806"],"confidence":"Medium","gaps":["In vivo target genes in retina not identified","Functional consequence of the splice insert on transcription not tested"]},{"year":2014,"claim":"Showed POU6F2/RPF-1 binds pituitary gene regulatory regions and transactivates Prop1 and Prrx2 (but not Prrx1) in progenitor cells, implicating it in pituitary developmental gene regulation.","evidence":"Yeast one-hybrid, luciferase reporter assays, in situ hybridization and RT-PCR in rat pituitary","pmids":["24804940"],"confidence":"Medium","gaps":["Direct in vivo promoter occupancy not confirmed by ChIP","Endogenous requirement for Prop1/Prrx2 regulation not tested by loss-of-function"]},{"year":2016,"claim":"Established human POU6F2/RPF-1 as a nuclear, sequence-specific transcriptional activator whose Ser-rich N-terminal region is required for DNA binding and whose induction suppresses proliferation and tumor growth, defining genome-wide candidate targets.","evidence":"Inducible transfection, EMSA with competition and deletion mutagenesis, reporter assays, ChIP-chip, transcriptome profiling, and xenograft assays","pmids":["27425396"],"confidence":"High","gaps":["Mechanism linking transcriptional targets to growth suppression not resolved","Direct vs. indirect status of the 217 candidate targets not validated individually"]},{"year":null,"claim":"It remains unresolved whether the sigma(70)-like RNA-binding activity of the ribosome-biogenesis RPF1 engages a specific pre-rRNA element, and the corpus does not reconcile the two distinct proteins sharing the RPF1 symbol.","evidence":"No direct RNA-substrate mapping or unifying analysis in the timeline","pmids":[],"confidence":"Low","gaps":["Direct RNA target of the sigma(70)-like domain unidentified","No structural/functional data on human RPF1 contacts within the 60S particle"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[7,8]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[8,9]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3,4,5]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[8,9]}],"complexes":["Nsa1-Rrp1-Rpf1-Mak16 pre-60S module","WDR74-RPF1-MAK16-RRP1 module"],"partners":["MAK16","RRP1","WDR74","NSA1","MTR4","KAP120","MAK5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H9Y2","full_name":"Ribosome production factor 1","aliases":["Brix domain-containing protein 5","Ribosome biogenesis protein RPF1"],"length_aa":349,"mass_kda":40.1,"function":"May be required for ribosome biogenesis","subcellular_location":"Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/Q9H9Y2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RPF1","classification":"Common Essential","n_dependent_lines":1120,"n_total_lines":1208,"dependency_fraction":0.9271523178807947},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RPF1","total_profiled":1310},"omim":[{"mim_id":"609062","title":"POU DOMAIN, CLASS 6, TRANSCRIPTION FACTOR 2; POU6F2","url":"https://www.omim.org/entry/609062"},{"mim_id":"602278","title":"NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWNREGULATED 4; NEDD4","url":"https://www.omim.org/entry/602278"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RPF1"},"hgnc":{"alias_symbol":[],"prev_symbol":["BXDC5"]},"alphafold":{"accession":"Q9H9Y2","domains":[{"cath_id":"3.40.50.10480","chopping":"107-340","consensus_level":"high","plddt":93.3451,"start":107,"end":340},{"cath_id":"1.20.5","chopping":"55-94","consensus_level":"high","plddt":88.8728,"start":55,"end":94}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H9Y2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H9Y2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H9Y2-F1-predicted_aligned_error_v6.png","plddt_mean":84.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RPF1","jax_strain_url":"https://www.jax.org/strain/search?query=RPF1"},"sequence":{"accession":"Q9H9Y2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H9Y2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H9Y2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H9Y2"}},"corpus_meta":[{"pmid":"10418975","id":"PMC_10418975","title":"Nuclear 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of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41231949","citation_count":0,"is_preprint":false},{"pmid":"40312777","id":"PMC_40312777","title":"Resuscitation-promoting factor diversity of Rhodococcus erythropolis KB1 and their promoting effect on bacterial growth and viable but non-culturable cell resuscitation.","date":"2025","source":"Letters in applied microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/40312777","citation_count":0,"is_preprint":false},{"pmid":"41743199","id":"PMC_41743199","title":"Pan-NLRome of Spinacia facilitates the rapid discovery of downy mildew resistance genes.","date":"2026","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/41743199","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":24887,"output_tokens":3137,"usd":0.060858,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10547,"output_tokens":3910,"usd":0.075243,"stage2_stop_reason":"end_turn"},"total_usd":0.136101,"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\": 2002,\n      \"finding\": \"RPF1 (yeast) belongs to the Imp4 superfamily of RNA-binding proteins that all possess the sigma(70)-like motif, a eukaryotic RNA binding domain with prokaryotic origins. Members of this superfamily associate with RNAs consistent with their distinct roles in ribosome biogenesis, and yeast Rpf1 is required for a specific stage of ribosome biogenesis.\",\n      \"method\": \"Bioinformatic identification of conserved sigma(70)-like RNA binding domain; functional requirement established by genetic analysis in yeast\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain identification combined with genetic requirement for ribosome biogenesis, single study\",\n      \"pmids\": [\"11864606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Kap120 functions as a nuclear import receptor for yeast Rpf1 (ribosome assembly factor). Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1 (via Kap120, Kap114, and Nmd5). Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus; overexpression of RPF1 suppresses the nuclear 60S accumulation in kap120 mutants, indicating Kap120 functions in nuclear import of Rpf1 rather than in ribosomal export directly.\",\n      \"method\": \"Two-hybrid screen, in vitro direct binding assay, Ran-GTP release assay, genetic epistasis (deletion mutants, overexpression suppression), nuclear accumulation phenotype assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal in vitro binding, Ran-GTP dissociation, genetic epistasis with overexpression suppression, multiple orthogonal methods in one study\",\n      \"pmids\": [\"16581791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Yeast Rpf1 is genetically linked to the DEAD-box RNA helicase Mak5, ribosomal protein Rpl14, and 60S biogenesis factors Ebp2 and Nop16 (synthetic lethality). Mak5 is associated with the Nsa1 pre-60S particle. These 'Mak5 cluster' factors, including Rpf1, are proposed to orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface (Rpl6, Rpl14, Rpl16 and rRNA expansion segments ES7L and ES39L).\",\n      \"method\": \"Synthetic lethal genetic screens with mak5 alleles; co-immunoprecipitation of Mak5 with the Nsa1 pre-60S particle\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — synthetic lethality screen and co-IP placing Rpf1 in a defined genetic/physical network, single lab\",\n      \"pmids\": [\"24312670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structures of early nucleolar pre-60S biogenesis intermediates at 3.3–4.5 Å resolution reveal that yeast Rpf1 is part of the Nsa1-Rrp1-Rpf1-Mak16 module, which stabilizes the solvent side of the 60S subunit during early nucleolar assembly. Rpf1 is mapped within six different assembly states, showing its position and integration order in the sequential folding pathway.\",\n      \"method\": \"Cryo-EM structural analysis of six pre-60S assembly intermediates; mapping of 25 assembly factors\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures at near-atomic resolution with multiple assembly states, direct mapping of Rpf1 in defined module, published in high-profile journal\",\n      \"pmids\": [\"29245012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Human RPF1 is required for accurate 60S ribosome biogenesis. siRNA-mediated knockdown of RPF1 in HEK293 cells significantly changed the pattern of RNA products derived from 47S pre-rRNA. RPF1 is associated with pre-60S particles (not pre-40S). RPF1 is not directly involved in pre-rRNA cleavage but rather helps pre-rRNA acquire the conformation favoring cleavage.\",\n      \"method\": \"shRNA/siRNA knockdown in HEK293 cells; pre-rRNA processing pattern analysis; pre-ribosomal particle association assay\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined pre-rRNA processing phenotype and particle association, single lab, single study\",\n      \"pmids\": [\"38391939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Human RPF1 functions as part of a pre-ribosomal WDR74 module (consisting of WDR74, RPF1, MAK16, and RRP1) that is mutually required for interactions with MTR4. Each component of the WDR74 module is mutually essential for the interaction of other members with MTR4, and all components are required for accurate cleavage of pre-rRNA during 60S ribosome biogenesis.\",\n      \"method\": \"Co-immunoprecipitation combined with mass spectrometry; functional knockdown analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP/MS identifying the complex and functional requirement established, single lab\",\n      \"pmids\": [\"39706051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Yeast Mak16 contains a [4Fe-4S] cluster and its interaction with Rpf1 is functionally dependent on Fe/S cluster integrity. Oxidative stress (H2O2) destabilizes Mak16 and disrupts its interaction with Rpf1 in vivo. Disruption of Fe/S cluster coordination in Mak16 impairs the Mak16-Rpf1 complex formation and decreases 25S rRNA level. Thus Rpf1 participates in a redox-sensitive step of early 60S maturation via its partner Mak16.\",\n      \"method\": \"In vivo and in vitro Fe/S cluster characterization; co-immunoprecipitation to detect Mak16-Rpf1 interaction under oxidative stress; rRNA level measurement after Fe/S cluster disruption mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (in vitro biochemistry, in vivo co-IP, mutagenesis, rRNA quantification) in a single rigorous study establishing the functional Mak16-Rpf1 interaction\",\n      \"pmids\": [\"41231949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"RPF-1 (POU6F2) encodes a POU-domain transcription factor expressed in retinal ganglion and amacrine cells. In vitro, the RPF-1 POU domain (without the alternatively spliced insert) binds to a consensus Oct-1 binding site, whereas the alternatively spliced isoform (with a 36 amino acid insert in the POU-specific domain DNA recognition helix) does not bind this site. RPF-1 protein first appears at e11 in mouse retina, localizing to neuroblasts that have recently migrated to the future ganglion cell layer.\",\n      \"method\": \"In vitro DNA binding assay (POU domain binding to Oct-1 consensus); cDNA/genomic cloning; in situ hybridization for subcellular/tissue localization\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct in vitro binding assay with mutagenesis-like isoform comparison, combined with developmental localization, single lab\",\n      \"pmids\": [\"8601806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Human RPF-1 (POU6F2) shows nuclear localization in HEK293 stable transfectants, elevated protein stability, and transactivation of promoters featuring POU consensus sites. RPF-1 induction leads to reduced cell proliferation and reduced in vivo tumor growth. Gel-shift competition assay demonstrated specificity of RPF-1 binding to consensus POU motifs, and showed that the Ser-rich region upstream of the POU domain is indispensable for DNA binding. Genome-wide ChIP-chip analysis predicted promoter occupancy consistent with transcriptome changes, identifying 217 candidate target genes.\",\n      \"method\": \"Stable inducible transfection; nuclear localization by direct imaging; transactivation reporter assay; gel-shift/EMSA with competition; deletion mutagenesis of Ser-rich region; ChIP-chip; transcriptome profiling; tumor xenograft growth assay\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (EMSA with mutagenesis, reporter assay, ChIP-chip, cellular and in vivo phenotype) in a single study\",\n      \"pmids\": [\"27425396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Rat/porcine RPF1 (POU6F2) protein binds specifically to the 5'-upstream region of porcine Fshβ gene, identified by Yeast One-Hybrid System. RPF1 shows regulatory (transactivation) activity for Prop1 and Prrx2 promoters but not for Prrx1 in reporter assays. RPF1 mRNA is expressed in the stem/progenitor cells of the rat pituitary primordium and decreases during pituitary development.\",\n      \"method\": \"Yeast one-hybrid system; luciferase reporter assay; in situ hybridization; RT-PCR\",\n      \"journal\": \"The Journal of reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct DNA binding by yeast one-hybrid plus reporter assay for transcriptional activity, single lab\",\n      \"pmids\": [\"24804940\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RPF1 encodes two functionally distinct proteins depending on context: in ribosome biogenesis (yeast Rpf1/human RPF1), it is a nucleolar assembly factor containing a sigma(70)-like RNA-binding domain that is imported into the nucleus via Kap120 (and parallel importins), associates with early pre-60S particles as part of the Nsa1-Rrp1-Rpf1-Mak16/WDR74 module to stabilize the solvent face of the 60S subunit, and is required for correct pre-rRNA processing and 25S rRNA accumulation; its partner Mak16 carries a redox-sensitive [4Fe-4S] cluster whose integrity is required for the Mak16-Rpf1 interaction. Separately, the human POU6F2/RPF-1 transcription factor uses its POU domain to bind octamer DNA consensus sites (with splice-isoform-dependent binding), transactivates target promoters including PROP1 and PRRX2 in developing pituitary and retina, requires its N-terminal Ser-rich region for DNA binding, and suppresses cell proliferation and tumor growth when induced.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"The RPF1 symbol maps to two unrelated proteins in this corpus, and the timeline coheres into two internally consistent mechanistic pictures. In ribosome biogenesis, yeast and human RPF1 is a nucleolar pre-60S assembly factor: yeast Rpf1 belongs to the Imp4 superfamily defined by a sigma(70)-like RNA-binding domain and is required for a specific stage of large-subunit biogenesis [#0]. It is delivered to the nucleus by the import receptor Kap120 (with parallel routes via Kap114 and Nmd5), as Kap120 binds Rpf1 directly and is released by Ran-GTP, and loss of either factor traps 60S particles in the nucleus [#1]. Within early nucleolar pre-60S intermediates, Rpf1 is an integral member of the Nsa1-Rrp1-Rpf1-Mak16 module that stabilizes the solvent-exposed face of the maturing 60S subunit [#3], a function that depends on the [4Fe-4S] cluster of its partner Mak16, whose oxidative disruption breaks the Mak16-Rpf1 interaction and lowers 25S rRNA levels, placing Rpf1 at a redox-sensitive step of 60S maturation [#6]. Human RPF1 is the conserved counterpart, associating with pre-60S (not pre-40S) particles and acting within a WDR74-RPF1-MAK16-RRP1 module whose members are mutually required for their interaction with the helicase MTR4 and for accurate pre-rRNA cleavage; RPF1 itself does not catalyze cleavage but promotes the pre-rRNA conformation that favors it [#4, #5]. Distinctly, the POU6F2/RPF-1 entries describe a POU-domain transcription factor: it binds octamer/POU consensus DNA in an isoform-dependent manner requiring an N-terminal Ser-rich region [#7, #8], transactivates developmental promoters including Prop1 and Prrx2 in pituitary progenitors [#9], and on induction localizes to the nucleus, suppresses proliferation, and reduces tumor xenograft growth [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that yeast Rpf1 is an RNA-binding protein of the Imp4/sigma(70)-like superfamily functionally required for a defined stage of ribosome biogenesis, defining its molecular class.\",\n      \"evidence\": \"Bioinformatic domain identification plus genetic requirement analysis in yeast\",\n      \"pmids\": [\"11864606\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not define which biogenesis substep or partner particle\", \"RNA target of the sigma(70)-like domain not directly mapped\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Answered how Rpf1 reaches the nucleolus, showing Kap120 is a direct import receptor (with parallel Kap114/Nmd5 routes) and that the nuclear 60S accumulation defect reflects failed Rpf1 import rather than a direct export role.\",\n      \"evidence\": \"Two-hybrid, in vitro binding, Ran-GTP release, and genetic epistasis/overexpression suppression in yeast\",\n      \"pmids\": [\"16581791\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Import signal on Rpf1 not delimited\", \"Relative contribution of the three parallel importins unquantified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed Rpf1 in a functional network with Mak5, Rpl14, Ebp2 and Nop16 implicated in shaping a eukaryote-specific 60S surface, linking it physically/genetically to the Nsa1 pre-60S particle.\",\n      \"evidence\": \"Synthetic lethal genetic screens and co-IP of Mak5 with the Nsa1 particle\",\n      \"pmids\": [\"24312670\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct Rpf1 contacts within the particle not resolved\", \"Genetic interactions do not prove direct binding\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved Rpf1's structural position by mapping it within the Nsa1-Rrp1-Rpf1-Mak16 module that stabilizes the solvent face of the 60S subunit across sequential early nucleolar intermediates.\",\n      \"evidence\": \"Cryo-EM of six pre-60S assembly states at 3.3-4.5 Å with assembly-factor mapping\",\n      \"pmids\": [\"29245012\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional consequence of each contact not tested by mutagenesis\", \"Order of module release during maturation not fully defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the role to human RPF1, showing it associates specifically with pre-60S particles and shapes pre-rRNA conformation to enable correct cleavage of 47S-derived precursors rather than catalyzing cleavage itself.\",\n      \"evidence\": \"siRNA/shRNA knockdown in HEK293, pre-rRNA processing analysis, particle association assays\",\n      \"pmids\": [\"38391939\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct RNA-binding by human RPF1 not demonstrated\", \"Single cell line, single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the human module as WDR74-RPF1-MAK16-RRP1 whose members are mutually required for engaging the helicase MTR4 and for accurate pre-rRNA cleavage, establishing interdependence within the module.\",\n      \"evidence\": \"Co-IP/mass spectrometry and functional knockdown\",\n      \"pmids\": [\"39706051\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct vs. indirect RPF1-MTR4 contact not distinguished\", \"Stoichiometry and assembly order of the module unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a redox control point in early 60S maturation by showing the Mak16 [4Fe-4S] cluster is required for the Mak16-Rpf1 interaction, with oxidative stress disrupting the complex and lowering 25S rRNA.\",\n      \"evidence\": \"In vitro/in vivo Fe/S characterization, co-IP under H2O2, and rRNA quantification after cluster-disrupting mutagenesis in yeast\",\n      \"pmids\": [\"41231949\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether Rpf1 itself senses redox is not addressed\", \"Physiological oxidative conditions that engage this switch not defined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Characterized the unrelated POU6F2/RPF-1 as a POU-domain transcription factor with isoform-dependent octamer DNA binding, expressed during retinal ganglion/amacrine cell development.\",\n      \"evidence\": \"In vitro DNA-binding assay with isoform comparison, cloning, and in situ hybridization in mouse retina\",\n      \"pmids\": [\"8601806\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"In vivo target genes in retina not identified\", \"Functional consequence of the splice insert on transcription not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed POU6F2/RPF-1 binds pituitary gene regulatory regions and transactivates Prop1 and Prrx2 (but not Prrx1) in progenitor cells, implicating it in pituitary developmental gene regulation.\",\n      \"evidence\": \"Yeast one-hybrid, luciferase reporter assays, in situ hybridization and RT-PCR in rat pituitary\",\n      \"pmids\": [\"24804940\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct in vivo promoter occupancy not confirmed by ChIP\", \"Endogenous requirement for Prop1/Prrx2 regulation not tested by loss-of-function\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established human POU6F2/RPF-1 as a nuclear, sequence-specific transcriptional activator whose Ser-rich N-terminal region is required for DNA binding and whose induction suppresses proliferation and tumor growth, defining genome-wide candidate targets.\",\n      \"evidence\": \"Inducible transfection, EMSA with competition and deletion mutagenesis, reporter assays, ChIP-chip, transcriptome profiling, and xenograft assays\",\n      \"pmids\": [\"27425396\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism linking transcriptional targets to growth suppression not resolved\", \"Direct vs. indirect status of the 217 candidate targets not validated individually\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved whether the sigma(70)-like RNA-binding activity of the ribosome-biogenesis RPF1 engages a specific pre-rRNA element, and the corpus does not reconcile the two distinct proteins sharing the RPF1 symbol.\",\n      \"evidence\": \"No direct RNA-substrate mapping or unifying analysis in the timeline\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct RNA target of the sigma(70)-like domain unidentified\", \"No structural/functional data on human RPF1 contacts within the 60S particle\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"complexes\": [\n      \"Nsa1-Rrp1-Rpf1-Mak16 pre-60S module\",\n      \"WDR74-RPF1-MAK16-RRP1 module\"\n    ],\n    \"partners\": [\n      \"MAK16\",\n      \"RRP1\",\n      \"WDR74\",\n      \"NSA1\",\n      \"MTR4\",\n      \"KAP120\",\n      \"MAK5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}