{"gene":"MDN1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2004,"finding":"Rea1 (MDN1) is required for maturation and nuclear export of the pre-60S ribosomal subunit; it localizes predominantly to the nucleoplasm and associates with a late pre-60S particle containing the Rix1 complex (Rix1, Ipi1, Ipi3). In vivo depletion causes defects in pre-rRNA processing and late pre-60S stability after ITS2 cleavage and prior to mature 5.8S rRNA generation, and results in nuclear accumulation of the large subunit reporter Rpl25-GFP.","method":"GAL-repressible and temperature-sensitive rea1 alleles; in vivo 60S export assay with Rpl25-GFP reporter; co-purification with Rix1 complex components","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic depletion, ts alleles, fluorescent export assay, co-purification), replicated across labs","pmids":["15528184"],"is_preprint":false},{"year":2018,"finding":"CryoEM structures of S. pombe Mdn1 (with AMPPNP at ~4 Å, or ATP+Rbin-1 at ~8 Å) reveal that its MIDAS domain is tethered to the hexameric AAA ring via an ~20 nm structured linker and a flexible ~500 aa Asp/Glu-rich motif. The MIDAS domain docks onto the AAA ring in a nucleotide state-specific manner, and conformational changes in the AAA ring are directly transmitted to the MIDAS domain to drive release of assembly factors from 60S precursors.","method":"Single-particle cryo-EM structure determination; chemical inhibitor (Rbin-1) treatment","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM structures with two nucleotide states, multiple orthogonal conditions","pmids":["30318141"],"is_preprint":false},{"year":2018,"finding":"CryoEM structures of S. cerevisiae Rea1 reveal the hexameric AAA+ ring architecture and identify an α-helical bundle within AAA2 as a major ATPase activity regulator that interferes with nucleotide-induced conformational changes creating a docking site for the MIDAS domain. The linker architecture extending from the AAA+ ring is also resolved and implicated in force generation.","method":"Single-particle cryo-EM structure determination","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple independent cryo-EM structures with functional interpretation, consistent with parallel Cell paper","pmids":["30460895"],"is_preprint":false},{"year":2019,"finding":"Crystal structures of the Rea1-MIDAS domain alone and in complex with the UBL domains of Rsa4 or Ytm1 show MIDAS-UBL complexes structurally similar to integrin α-subunit/ligand interactions. A loop insert in MIDAS functions as an NLS and activates the mechanochemical Rea1 cycle, while an additional β-hairpin anchors the UBL domain substrate. These structures establish that the MIDAS domain physically engages UBL-domain-containing assembly factors for their extraction.","method":"X-ray crystallography of MIDAS domain alone and in complex with Rsa4-UBL and Ytm1-UBL","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution crystal structures with two different substrates, multiple independent complexes","pmids":["31296859"],"is_preprint":false},{"year":2020,"finding":"Mdn1's MIDAS domain can dock onto the AAA ring in a bimolecular (intermolecular) manner and this docking reduces ATPase activity. Tethering the MIDAS domain to the AAA ring via the linker prevents, rather than promotes, MIDAS docking in the absence of inducing signals (preribosome binding or chemical inhibitor treatment), revealing long-range intramolecular allostery.","method":"Chemical probes, single-particle electron microscopy, native mass spectrometry; domain-truncation constructs tested for ATPase activity and MIDAS-ring interaction","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — three orthogonal methods (EM, native MS, biochemical ATPase assay), single lab but multiple complementary approaches","pmids":["32694211"],"is_preprint":false},{"year":2022,"finding":"Mdn1's MIDAS domain forms catch bonds with the UBL domains of both Rsa4 and Ytm1: forces up to ~4 pN (consistent with AAA ATPase force output) enhance MIDAS binding lifetime up to 10-fold, while higher forces accelerate dissociation. This catch-bond mechanoregulation is proposed to underlie switching between strongly and weakly bound states during the Mdn1 enzymatic cycle.","method":"Optical tweezers force spectroscopy measuring force-dependence of MIDAS-UBL binding lifetime for both Rsa4 and Ytm1","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct single-molecule force measurement with two substrates in parallel, single lab","pmids":["35147499"],"is_preprint":false},{"year":2023,"finding":"Rea1 removes the Ytm1-Erb1 heterodimer from nucleolar pre-60S particles in a step that is concurrent with Spb4 helicase-mediated restructuring of 25S rRNA helices H62 and H63/H63a. In vitro maturation assays with purified Rea1 and the pentameric Rix1 complex plus ATP, combined with cryo-EM, show that Rea1 ATPase activity drives large-scale remodeling and release of a network of assembly factors after rRNA restructuring.","method":"In vitro maturation assay with purified Rea1, Rix1 complex, and pre-60S particles; cryo-EM structural analysis of Spb4-enriched pre-60S intermediates","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution in vitro with purified components, cryo-EM structural validation, single lab","pmids":["36929751"],"is_preprint":false},{"year":2024,"finding":"The Rea1 linker domain (stem, middle, top subdomains) is essential for assembly factor removal. It undergoes nucleotide-independent and nucleotide-dependent remodeling steps: ATP hydrolysis is required for the linker to engage with the AAA+ ring and subsequently with the AAA-ring-docked MIDAS domain, enabling direct force transmission from the linker top to MIDAS for assembly factor extraction.","method":"cryo-EM structural analysis of Rea1 in different nucleotide states; functional assays demonstrating linker requirement for assembly factor removal","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — cryo-EM structures combined with functional assays showing linker necessity, single lab, multiple nucleotide states","pmids":["39604383"],"is_preprint":false},{"year":2025,"finding":"Depletion of MDN1 in HEK293T cells results in decreased levels of RNAs involved in ribosome biogenesis (strongest effect among Ki-67, GNL2, and MDN1), decreased transcripts for mitochondrial respiration, and strong culture acidification. MDN1 is recruited to the nucleolar periphery via interaction with Ki-67 and GNL2, and its depletion alters nucleolar protein and chromatin localization.","method":"siRNA depletion; RNA-seq; confocal microscopy; biochemical co-immunoprecipitation/protein-protein interaction data in HEK293T cells","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, phenotypic RNA-seq and microscopy readouts with limited mechanistic depth for MDN1 specifically","pmids":["bio_10.1101_2025.02.13.638155"],"is_preprint":true}],"current_model":"MDN1/Rea1 is a massive (~5000 aa) AAA+ ATPase mechanoenzyme that drives ribosome biogenesis by using ATP hydrolysis to mechanically extract UBL-domain-containing assembly factors (Ytm1, Rsa4) from pre-60S ribosomal particles via its C-terminal MIDAS domain, which forms catch bonds with these substrates; conformational changes in the hexameric AAA ring are transmitted through an ~20 nm structured linker to the MIDAS domain in a nucleotide-state-dependent docking mechanism, and intramolecular tethering of MIDAS to the AAA ring normally suppresses docking until preribosome engagement triggers the mechanochemical cycle, ultimately enabling nuclear export of the mature 60S subunit."},"narrative":{"mechanistic_narrative":"MDN1 (Rea1) is a giant AAA+ ATPase mechanoenzyme that drives the late nucleoplasmic maturation and nuclear export of the pre-60S large ribosomal subunit [PMID:15528184]. It engages late pre-60S particles in association with the Rix1 complex and, through ATP-driven remodeling, mechanically extracts UBL-domain-containing assembly factors such as Ytm1 (with its partner Erb1) and Rsa4 from the maturing particle, a remodeling step coordinated with helicase-mediated restructuring of 25S rRNA helices H62/H63 [PMID:15528184, PMID:36929751]. Substrate extraction is executed by a C-terminal MIDAS domain that physically clamps the UBL domains of Rsa4 and Ytm1 in an integrin-like interaction and forms catch bonds with them, so that piconewton-scale forces consistent with AAA+ ATPase output stabilize binding before higher forces trigger release [PMID:31296859, PMID:35147499]. The MIDAS domain is connected to the hexameric AAA+ ring by an ~20 nm structured linker; conformational changes within the ring are propagated through this linker to dock and undock MIDAS in a nucleotide-state-dependent manner, with ATP hydrolysis required for the linker to engage the ring and then the docked MIDAS domain to transmit force [PMID:30318141, PMID:30460895, PMID:39604383]. Intramolecular tethering of MIDAS to the AAA ring normally suppresses docking until preribosome engagement or chemical inducers trigger the mechanochemical cycle, establishing a long-range autoinhibitory allostery, and an α-helical bundle within AAA2 regulates ATPase activity by controlling formation of the MIDAS docking site [PMID:30460895, PMID:32694211].","teleology":[{"year":2004,"claim":"Established that Rea1/MDN1 is a dedicated late-acting factor for pre-60S maturation and export rather than a general housekeeping ATPase, defining the cellular process it serves.","evidence":"GAL-repressible and ts rea1 alleles, Rpl25-GFP export reporter, and co-purification with the Rix1 complex in yeast","pmids":["15528184"],"confidence":"High","gaps":["Did not reveal the molecular mechanism of action or direct substrates","Recruitment to the particle beyond Rix1 association undefined"]},{"year":2018,"claim":"Resolved the architecture coupling the AAA+ motor to the substrate-engaging MIDAS domain, answering how a single enzyme converts nucleotide cycling into long-range mechanical output.","evidence":"Single-particle cryo-EM of S. pombe Mdn1 and S. cerevisiae Rea1 in distinct nucleotide states, including the ~20 nm linker and an AAA2 α-helical regulatory bundle","pmids":["30318141","30460895"],"confidence":"High","gaps":["Atomic detail of MIDAS-substrate engagement not resolved","Order and directionality of force transmission inferred but not directly demonstrated"]},{"year":2019,"claim":"Defined the structural basis for substrate recognition, showing MIDAS directly clamps the UBL domains of the assembly factors it extracts.","evidence":"X-ray crystallography of the Rea1 MIDAS domain alone and bound to Rsa4-UBL and Ytm1-UBL, revealing integrin-like contacts, an NLS loop, and an anchoring β-hairpin","pmids":["31296859"],"confidence":"High","gaps":["Static structures do not show the dynamics of extraction","Affinity modulation during the catalytic cycle not addressed"]},{"year":2020,"claim":"Showed that MIDOS-ring docking is autoinhibitory, reframing the linker tether as a suppressor of premature engagement rather than a simple connector.","evidence":"Chemical probes, single-particle EM, native mass spectrometry, and domain-truncation ATPase assays comparing intramolecular vs intermolecular MIDAS docking","pmids":["32694211"],"confidence":"High","gaps":["Single-lab; in vivo relevance of intermolecular docking not established","Precise signal from preribosome that relieves autoinhibition unidentified"]},{"year":2022,"claim":"Demonstrated that MIDAS-UBL bonds are force-activated catch bonds, providing the mechanistic logic for switching between gripping and releasing substrate during the enzymatic cycle.","evidence":"Optical tweezers force spectroscopy of MIDAS binding lifetimes to Rsa4 and Ytm1 across a force range","pmids":["35147499"],"confidence":"High","gaps":["Force values inferred from isolated domains rather than measured on intact enzyme on particles","Single lab"]},{"year":2023,"claim":"Reconstituted Rea1-driven remodeling in vitro and placed factor removal in temporal context with rRNA restructuring, linking motor action to a defined maturation transition.","evidence":"In vitro maturation assays with purified Rea1, the Rix1 complex, ATP, and pre-60S particles plus cryo-EM of Spb4-enriched intermediates","pmids":["36929751"],"confidence":"High","gaps":["Coordination between Spb4 helicase and Rea1 mechanistically unresolved","Single lab"]},{"year":2024,"claim":"Dissected the linker as the active force-transmitting element, showing ATP hydrolysis sequentially licenses linker-ring and linker-MIDAS engagement.","evidence":"Cryo-EM of Rea1 in multiple nucleotide states with functional assays showing the stem/middle/top linker subdomains are required for assembly factor removal","pmids":["39604383"],"confidence":"High","gaps":["Quantitative force per cycle not measured on the full machine","Single lab"]},{"year":2025,"claim":"Extended the yeast-defined role to human cells, proposing nucleolar-periphery recruitment via Ki-67 and GNL2 and a role in ribosome-biogenesis transcript levels.","evidence":"siRNA depletion, RNA-seq, confocal microscopy, and co-IP in HEK293T cells (bioRxiv preprint)","pmids":["bio_10.1101_2025.02.13.638155"],"confidence":"Low","gaps":["Preprint, single lab with limited mechanistic depth for MDN1","Ki-67/GNL2 interaction not validated reciprocally or structurally","Direct enzymatic role in human pre-60S not demonstrated"]},{"year":null,"claim":"How the preribosome triggers relief of MIDAS autoinhibition and how the full mechanochemical cycle is coordinated with helicases and the Rix1 complex on native particles remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["Trigger signal coupling particle binding to docking unidentified","No integrated structure of the actively extracting machine on pre-60S","Human pathway largely uncharacterized in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[1,2,4,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,5,6]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[3,5]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[6,8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0]}],"complexes":["Rix1 complex (associated)"],"partners":["RSA4","YTM1","ERB1","SPB4","RIX1","KI67","GNL2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NU22","full_name":"Midasin","aliases":["Dynein-related AAA-ATPase MDN1","MIDAS-containing protein"],"length_aa":5596,"mass_kda":632.8,"function":"Nuclear chaperone required for maturation and nuclear export of pre-60S ribosome subunits (PubMed:27814492). Functions at successive maturation steps to remove ribosomal factors at critical transition points, first driving the exit of early pre-60S particles from the nucleolus and then driving late pre-60S particles from the nucleus (By similarity). At an early stage in 60S maturation, mediates the dissociation of the PeBoW complex (PES1-BOP1-WDR12) from early pre-60S particles, rendering them competent for export from the nucleolus to the nucleoplasm (By similarity). Subsequently recruited to the nucleoplasmic particles through interaction with SUMO-conjugated PELP1 complex (PubMed:27814492). This binding is only possible if the 5S RNP at the central protuberance has undergone the rotation to complete its maturation (By similarity)","subcellular_location":"Nucleus, nucleolus; Nucleus, nucleoplasm; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9NU22/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MDN1","classification":"Common Essential","n_dependent_lines":1196,"n_total_lines":1208,"dependency_fraction":0.9900662251655629},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MDN1","total_profiled":1310},"omim":[{"mim_id":"618200","title":"MIDASIN AAA ATPase 1; MDN1","url":"https://www.omim.org/entry/618200"},{"mim_id":"616717","title":"TESTIS-EXPRESSED GENE 10; TEX10","url":"https://www.omim.org/entry/616717"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Intermediate filaments","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MDN1"},"hgnc":{"alias_symbol":["KIAA0301","Rea1"],"prev_symbol":[]},"alphafold":{"accession":"Q9NU22","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NU22","model_url":"","pae_url":"","plddt_mean":null},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MDN1","jax_strain_url":"https://www.jax.org/strain/search?query=MDN1"},"sequence":{"accession":"Q9NU22","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NU22.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NU22/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NU22"}},"corpus_meta":[{"pmid":"15528184","id":"PMC_15528184","title":"Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNA processing and nuclear export of 60 S subunits.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15528184","citation_count":62,"is_preprint":false},{"pmid":"30318141","id":"PMC_30318141","title":"Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis.","date":"2018","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/30318141","citation_count":45,"is_preprint":false},{"pmid":"30460895","id":"PMC_30460895","title":"The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1.","date":"2018","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/30460895","citation_count":25,"is_preprint":false},{"pmid":"31296859","id":"PMC_31296859","title":"Crystal structures of Rea1-MIDAS bound to its ribosome assembly factor ligands resembling integrin-ligand-type complexes.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31296859","citation_count":19,"is_preprint":false},{"pmid":"36929751","id":"PMC_36929751","title":"Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase.","date":"2023","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/36929751","citation_count":11,"is_preprint":false},{"pmid":"35147499","id":"PMC_35147499","title":"The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates.","date":"2022","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/35147499","citation_count":10,"is_preprint":false},{"pmid":"32694211","id":"PMC_32694211","title":"Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32694211","citation_count":9,"is_preprint":false},{"pmid":"40217384","id":"PMC_40217384","title":"MDN1 variants cause susceptibility to epilepsy : For the China Epilepsy Gene 1.0 Project.","date":"2025","source":"Acta epileptologica","url":"https://pubmed.ncbi.nlm.nih.gov/40217384","citation_count":3,"is_preprint":false},{"pmid":"19737511","id":"PMC_19737511","title":"The Rea1 tadpole loses its tail.","date":"2009","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/19737511","citation_count":2,"is_preprint":false},{"pmid":"39604383","id":"PMC_39604383","title":"Remodelling of Rea1 linker domain drives the removal of assembly factors from pre-ribosomal particles.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39604383","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.02.13.638155","title":"The roles of MKI67, GNL2, and MDN1 in Ribosome biogenesis and Transcriptome regulation in the Neuronal Lineage cell line HEK293T","date":"2025-02-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.13.638155","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6949,"output_tokens":2550,"usd":0.029548,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9728,"output_tokens":3031,"usd":0.062207,"stage2_stop_reason":"end_turn"},"total_usd":0.091755,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"Rea1 (MDN1) is required for maturation and nuclear export of the pre-60S ribosomal subunit; it localizes predominantly to the nucleoplasm and associates with a late pre-60S particle containing the Rix1 complex (Rix1, Ipi1, Ipi3). In vivo depletion causes defects in pre-rRNA processing and late pre-60S stability after ITS2 cleavage and prior to mature 5.8S rRNA generation, and results in nuclear accumulation of the large subunit reporter Rpl25-GFP.\",\n      \"method\": \"GAL-repressible and temperature-sensitive rea1 alleles; in vivo 60S export assay with Rpl25-GFP reporter; co-purification with Rix1 complex components\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic depletion, ts alleles, fluorescent export assay, co-purification), replicated across labs\",\n      \"pmids\": [\"15528184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CryoEM structures of S. pombe Mdn1 (with AMPPNP at ~4 Å, or ATP+Rbin-1 at ~8 Å) reveal that its MIDAS domain is tethered to the hexameric AAA ring via an ~20 nm structured linker and a flexible ~500 aa Asp/Glu-rich motif. The MIDAS domain docks onto the AAA ring in a nucleotide state-specific manner, and conformational changes in the AAA ring are directly transmitted to the MIDAS domain to drive release of assembly factors from 60S precursors.\",\n      \"method\": \"Single-particle cryo-EM structure determination; chemical inhibitor (Rbin-1) treatment\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM structures with two nucleotide states, multiple orthogonal conditions\",\n      \"pmids\": [\"30318141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CryoEM structures of S. cerevisiae Rea1 reveal the hexameric AAA+ ring architecture and identify an α-helical bundle within AAA2 as a major ATPase activity regulator that interferes with nucleotide-induced conformational changes creating a docking site for the MIDAS domain. The linker architecture extending from the AAA+ ring is also resolved and implicated in force generation.\",\n      \"method\": \"Single-particle cryo-EM structure determination\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple independent cryo-EM structures with functional interpretation, consistent with parallel Cell paper\",\n      \"pmids\": [\"30460895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structures of the Rea1-MIDAS domain alone and in complex with the UBL domains of Rsa4 or Ytm1 show MIDAS-UBL complexes structurally similar to integrin α-subunit/ligand interactions. A loop insert in MIDAS functions as an NLS and activates the mechanochemical Rea1 cycle, while an additional β-hairpin anchors the UBL domain substrate. These structures establish that the MIDAS domain physically engages UBL-domain-containing assembly factors for their extraction.\",\n      \"method\": \"X-ray crystallography of MIDAS domain alone and in complex with Rsa4-UBL and Ytm1-UBL\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution crystal structures with two different substrates, multiple independent complexes\",\n      \"pmids\": [\"31296859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mdn1's MIDAS domain can dock onto the AAA ring in a bimolecular (intermolecular) manner and this docking reduces ATPase activity. Tethering the MIDAS domain to the AAA ring via the linker prevents, rather than promotes, MIDAS docking in the absence of inducing signals (preribosome binding or chemical inhibitor treatment), revealing long-range intramolecular allostery.\",\n      \"method\": \"Chemical probes, single-particle electron microscopy, native mass spectrometry; domain-truncation constructs tested for ATPase activity and MIDAS-ring interaction\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — three orthogonal methods (EM, native MS, biochemical ATPase assay), single lab but multiple complementary approaches\",\n      \"pmids\": [\"32694211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Mdn1's MIDAS domain forms catch bonds with the UBL domains of both Rsa4 and Ytm1: forces up to ~4 pN (consistent with AAA ATPase force output) enhance MIDAS binding lifetime up to 10-fold, while higher forces accelerate dissociation. This catch-bond mechanoregulation is proposed to underlie switching between strongly and weakly bound states during the Mdn1 enzymatic cycle.\",\n      \"method\": \"Optical tweezers force spectroscopy measuring force-dependence of MIDAS-UBL binding lifetime for both Rsa4 and Ytm1\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct single-molecule force measurement with two substrates in parallel, single lab\",\n      \"pmids\": [\"35147499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Rea1 removes the Ytm1-Erb1 heterodimer from nucleolar pre-60S particles in a step that is concurrent with Spb4 helicase-mediated restructuring of 25S rRNA helices H62 and H63/H63a. In vitro maturation assays with purified Rea1 and the pentameric Rix1 complex plus ATP, combined with cryo-EM, show that Rea1 ATPase activity drives large-scale remodeling and release of a network of assembly factors after rRNA restructuring.\",\n      \"method\": \"In vitro maturation assay with purified Rea1, Rix1 complex, and pre-60S particles; cryo-EM structural analysis of Spb4-enriched pre-60S intermediates\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution in vitro with purified components, cryo-EM structural validation, single lab\",\n      \"pmids\": [\"36929751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The Rea1 linker domain (stem, middle, top subdomains) is essential for assembly factor removal. It undergoes nucleotide-independent and nucleotide-dependent remodeling steps: ATP hydrolysis is required for the linker to engage with the AAA+ ring and subsequently with the AAA-ring-docked MIDAS domain, enabling direct force transmission from the linker top to MIDAS for assembly factor extraction.\",\n      \"method\": \"cryo-EM structural analysis of Rea1 in different nucleotide states; functional assays demonstrating linker requirement for assembly factor removal\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cryo-EM structures combined with functional assays showing linker necessity, single lab, multiple nucleotide states\",\n      \"pmids\": [\"39604383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Depletion of MDN1 in HEK293T cells results in decreased levels of RNAs involved in ribosome biogenesis (strongest effect among Ki-67, GNL2, and MDN1), decreased transcripts for mitochondrial respiration, and strong culture acidification. MDN1 is recruited to the nucleolar periphery via interaction with Ki-67 and GNL2, and its depletion alters nucleolar protein and chromatin localization.\",\n      \"method\": \"siRNA depletion; RNA-seq; confocal microscopy; biochemical co-immunoprecipitation/protein-protein interaction data in HEK293T cells\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, phenotypic RNA-seq and microscopy readouts with limited mechanistic depth for MDN1 specifically\",\n      \"pmids\": [\"bio_10.1101_2025.02.13.638155\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MDN1/Rea1 is a massive (~5000 aa) AAA+ ATPase mechanoenzyme that drives ribosome biogenesis by using ATP hydrolysis to mechanically extract UBL-domain-containing assembly factors (Ytm1, Rsa4) from pre-60S ribosomal particles via its C-terminal MIDAS domain, which forms catch bonds with these substrates; conformational changes in the hexameric AAA ring are transmitted through an ~20 nm structured linker to the MIDAS domain in a nucleotide-state-dependent docking mechanism, and intramolecular tethering of MIDAS to the AAA ring normally suppresses docking until preribosome engagement triggers the mechanochemical cycle, ultimately enabling nuclear export of the mature 60S subunit.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MDN1 (Rea1) is a giant AAA+ ATPase mechanoenzyme that drives the late nucleoplasmic maturation and nuclear export of the pre-60S large ribosomal subunit [#0]. It engages late pre-60S particles in association with the Rix1 complex and, through ATP-driven remodeling, mechanically extracts UBL-domain-containing assembly factors such as Ytm1 (with its partner Erb1) and Rsa4 from the maturing particle, a remodeling step coordinated with helicase-mediated restructuring of 25S rRNA helices H62/H63 [#0, #6]. Substrate extraction is executed by a C-terminal MIDAS domain that physically clamps the UBL domains of Rsa4 and Ytm1 in an integrin-like interaction and forms catch bonds with them, so that piconewton-scale forces consistent with AAA+ ATPase output stabilize binding before higher forces trigger release [#3, #5]. The MIDAS domain is connected to the hexameric AAA+ ring by an ~20 nm structured linker; conformational changes within the ring are propagated through this linker to dock and undock MIDAS in a nucleotide-state-dependent manner, with ATP hydrolysis required for the linker to engage the ring and then the docked MIDAS domain to transmit force [#1, #2, #7]. Intramolecular tethering of MIDAS to the AAA ring normally suppresses docking until preribosome engagement or chemical inducers trigger the mechanochemical cycle, establishing a long-range autoinhibitory allostery, and an α-helical bundle within AAA2 regulates ATPase activity by controlling formation of the MIDAS docking site [#2, #4].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that Rea1/MDN1 is a dedicated late-acting factor for pre-60S maturation and export rather than a general housekeeping ATPase, defining the cellular process it serves.\",\n      \"evidence\": \"GAL-repressible and ts rea1 alleles, Rpl25-GFP export reporter, and co-purification with the Rix1 complex in yeast\",\n      \"pmids\": [\"15528184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not reveal the molecular mechanism of action or direct substrates\", \"Recruitment to the particle beyond Rix1 association undefined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved the architecture coupling the AAA+ motor to the substrate-engaging MIDAS domain, answering how a single enzyme converts nucleotide cycling into long-range mechanical output.\",\n      \"evidence\": \"Single-particle cryo-EM of S. pombe Mdn1 and S. cerevisiae Rea1 in distinct nucleotide states, including the ~20 nm linker and an AAA2 α-helical regulatory bundle\",\n      \"pmids\": [\"30318141\", \"30460895\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic detail of MIDAS-substrate engagement not resolved\", \"Order and directionality of force transmission inferred but not directly demonstrated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the structural basis for substrate recognition, showing MIDAS directly clamps the UBL domains of the assembly factors it extracts.\",\n      \"evidence\": \"X-ray crystallography of the Rea1 MIDAS domain alone and bound to Rsa4-UBL and Ytm1-UBL, revealing integrin-like contacts, an NLS loop, and an anchoring β-hairpin\",\n      \"pmids\": [\"31296859\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Static structures do not show the dynamics of extraction\", \"Affinity modulation during the catalytic cycle not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed that MIDOS-ring docking is autoinhibitory, reframing the linker tether as a suppressor of premature engagement rather than a simple connector.\",\n      \"evidence\": \"Chemical probes, single-particle EM, native mass spectrometry, and domain-truncation ATPase assays comparing intramolecular vs intermolecular MIDAS docking\",\n      \"pmids\": [\"32694211\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single-lab; in vivo relevance of intermolecular docking not established\", \"Precise signal from preribosome that relieves autoinhibition unidentified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that MIDAS-UBL bonds are force-activated catch bonds, providing the mechanistic logic for switching between gripping and releasing substrate during the enzymatic cycle.\",\n      \"evidence\": \"Optical tweezers force spectroscopy of MIDAS binding lifetimes to Rsa4 and Ytm1 across a force range\",\n      \"pmids\": [\"35147499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Force values inferred from isolated domains rather than measured on intact enzyme on particles\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Reconstituted Rea1-driven remodeling in vitro and placed factor removal in temporal context with rRNA restructuring, linking motor action to a defined maturation transition.\",\n      \"evidence\": \"In vitro maturation assays with purified Rea1, the Rix1 complex, ATP, and pre-60S particles plus cryo-EM of Spb4-enriched intermediates\",\n      \"pmids\": [\"36929751\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination between Spb4 helicase and Rea1 mechanistically unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Dissected the linker as the active force-transmitting element, showing ATP hydrolysis sequentially licenses linker-ring and linker-MIDAS engagement.\",\n      \"evidence\": \"Cryo-EM of Rea1 in multiple nucleotide states with functional assays showing the stem/middle/top linker subdomains are required for assembly factor removal\",\n      \"pmids\": [\"39604383\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative force per cycle not measured on the full machine\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the yeast-defined role to human cells, proposing nucleolar-periphery recruitment via Ki-67 and GNL2 and a role in ribosome-biogenesis transcript levels.\",\n      \"evidence\": \"siRNA depletion, RNA-seq, confocal microscopy, and co-IP in HEK293T cells (bioRxiv preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.02.13.638155\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, single lab with limited mechanistic depth for MDN1\", \"Ki-67/GNL2 interaction not validated reciprocally or structurally\", \"Direct enzymatic role in human pre-60S not demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the preribosome triggers relief of MIDAS autoinhibition and how the full mechanochemical cycle is coordinated with helicases and the Rix1 complex on native particles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger signal coupling particle binding to docking unidentified\", \"No integrated structure of the actively extracting machine on pre-60S\", \"Human pathway largely uncharacterized in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [1, 2, 4, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 5, 6]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [6, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"Rix1 complex (associated)\"],\n    \"partners\": [\"Rsa4\", \"Ytm1\", \"Erb1\", \"Spb4\", \"Rix1\", \"KI67\", \"GNL2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}