{"gene":"MAK16","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1988,"finding":"MAK16 encodes a 306-amino acid nuclear protein (Mr 35,694) with two nuclear localization signal sequences; MAK16-LacZ fusion proteins containing one NLS entered the nucleus as determined by subcellular fractionation, while unfused beta-galactosidase did not. Loss-of-function (mak16-1) causes G1 arrest at non-permissive temperature, with cells remaining mating competent, placing MAK16 function in G1 cell cycle progression.","method":"Subcellular fractionation of MAK16-LacZ fusion proteins; cell cycle staging by mating competence assay; DNA sequencing to identify NLS motifs","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct fractionation experiment demonstrating nuclear localization, combined with cell-cycle arrest phenotype from loss-of-function; single lab, two orthogonal methods","pmids":["3045810"],"is_preprint":false},{"year":1995,"finding":"mak16 mutants have decreased free 60S ribosomal subunits, as shown by polysome profile analysis; supplying the normal MAK16 gene restored free 60S subunit levels, L-A copy number, and L-A coat protein to wild-type levels, establishing that MAK16 function is required for 60S ribosomal subunit supply.","method":"Sucrose gradient polysome profiling; complementation with single-copy MAK16 gene","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — polysome profiling plus genetic complementation, single lab, two orthogonal methods","pmids":["7739558"],"is_preprint":false},{"year":2000,"finding":"The Schistosoma mansoni MAK16 homolog (SmMAK16) is phosphorylated in vitro by human protein kinase CK2. GFP fusions of SmMAK16 localize exclusively to nucleoli (not nuclei) in COS-7 cells, and the yeast and C. elegans homologs similarly direct nucleolar accumulation, establishing nucleolar targeting as a conserved feature of MAK16 family proteins and suggesting a role in 60S subunit biogenesis.","method":"In vitro phosphorylation assay with recombinant CK2; transient transfection of GFP fusion constructs in COS-7 cells; fluorescence microscopy","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct live-cell imaging of GFP fusions plus in vitro kinase assay; single lab, two orthogonal methods","pmids":["10838225"],"is_preprint":false},{"year":2006,"finding":"Mak16p is required for the stability of the 27SB precursor rRNA in S. cerevisiae: at non-permissive temperature, mak16-1 cells show a dramatic decrease in 27SB pre-rRNA levels, leading to loss of mature 25S and 5.8S rRNAs, while 18S and 5S are only moderately affected. The mutant Mak16-1p protein was still associated with the 66S pre-ribosomal complex, indicating that mere association with the 66S pre-RNP is insufficient for function.","method":"Northern blot analysis of rRNA processing intermediates in temperature-sensitive mak16-1 yeast; sucrose density gradient fractionation","journal":"Yeast (Chichester, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Northern blot rRNA processing combined with gradient fractionation; single lab, two orthogonal methods","pmids":["16710831"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structures of early nucleolar 60S biogenesis intermediates at 3.3–4.5 Å resolution reveal that Mak16 functions as part of the Nsa1-Rrp1-Rpf1-Mak16 module, which stabilizes the solvent side of the 60S subunit during early nucleolar pre-60S assembly. The structural snapshots show Mak16 mapped to specific assembly states in the sequential folding pathway.","method":"Cryo-electron microscopy (cryo-EM) of purified early 60S biogenesis intermediates from S. cerevisiae; structural modeling at 3.3–4.5 Å resolution","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with functional pathway placement; replicated in independent structural studies","pmids":["29245012"],"is_preprint":false},{"year":2022,"finding":"Deletion of eIF4G1 in S. cerevisiae causes abnormal retention of Mak16 (along with Ssf1, Rrp15, Rrp14) on the pre-60S complex, impairing polypeptide exit tunnel (PET) maturation and 27S processing, placing Mak16 in a pathway downstream of eIF4G1 during pre-60S maturation.","method":"Mass spectrometry of purified pre-60S complexes from eIF4G1 deletion yeast; genetic interaction analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry of purified complexes combined with genetic deletion; single lab","pmids":["35615984"],"is_preprint":false},{"year":2024,"finding":"In human cells, MAK16 functions as part of a pre-ribosomal WDR74 module (comprising WDR74, RPF1, MAK16, and RRP1) identified by co-immunoprecipitation coupled with mass spectrometry. Each component of this module is mutually required for interaction of other members with the RNA helicase MTR4, and all components are required for accurate cleavage of pre-rRNA during 60S biogenesis.","method":"Co-immunoprecipitation combined with mass spectrometry; functional knockdown experiments monitoring pre-rRNA processing","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS plus functional knockdown with defined pre-rRNA processing readout; single lab","pmids":["39706051"],"is_preprint":false},{"year":2025,"finding":"Mak16 (yeast and human) contains a redox-active [4Fe-4S]2+/1+ cluster with midpoint potential below -500 mV, identified by both in vivo and in vitro methods. Oxidative stress (H2O2) causes [3Fe-4S]1+ cluster formation, destabilizes Mak16, and disrupts its interaction with Rpf1 in vivo. Disruption of Fe/S cluster coordination by mutagenesis destabilized Mak16, impaired Mak16-Rpf1 complex formation, and decreased 25S rRNA levels, establishing the Fe/S cluster as essential for Mak16 function in 60S ribosome biogenesis and as a potential sensor for redox imbalance.","method":"In vitro Fe/S cluster characterization (EPR spectroscopy, electrochemistry); in vivo H2O2 treatment; mutagenesis of Fe/S cluster coordination residues; co-immunoprecipitation of Mak16-Rpf1 complex; Northern blot for 25S rRNA levels","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution and characterization of Fe/S cluster combined with mutagenesis, in vivo functional validation, and structural correlation; multiple orthogonal methods in single rigorous study","pmids":["41231949"],"is_preprint":false}],"current_model":"MAK16 is an essential nucleolar protein that, as part of the Nsa1/Rrp1/Rpf1/Mak16 module, stabilizes the solvent face of early pre-60S ribosomal intermediates and is required for 27SB pre-rRNA stability and maturation of 25S and 5.8S rRNAs; its function critically depends on a redox-active [4Fe-4S] cluster that mediates its interaction with partner Rpf1 and is destabilized by oxidative stress, linking ribosome assembly to cellular redox status."},"narrative":{"mechanistic_narrative":"MAK16 is an essential, conserved nucleolar protein required for biogenesis of the large (60S) ribosomal subunit [PMID:7739558, PMID:10838225]. Within early nucleolar pre-60S intermediates it acts as a component of the Nsa1-Rrp1-Rpf1-Mak16 module that stabilizes the solvent face of the assembling subunit, with cryo-EM placing it at defined states of the sequential rRNA folding pathway [PMID:29245012]; an equivalent human pre-ribosomal module (WDR74-RPF1-MAK16-RRP1) couples to the RNA helicase MTR4 and is collectively required for accurate pre-rRNA cleavage during 60S biogenesis [PMID:39706051]. Functionally, Mak16 is needed for stability of the 27SB pre-rRNA and hence for maturation of mature 25S and 5.8S rRNAs, and its proper release from the pre-60S particle is governed by upstream factors such as eIF4G1 [PMID:16710831, PMID:35615984]. Mak16 harbors a redox-active [4Fe-4S]2+/1+ cluster (midpoint potential below -500 mV) that is essential for its stability and for its interaction with the partner Rpf1; oxidative stress converts it to a [3Fe-4S]1+ form, destabilizes the protein, disrupts the Mak16-Rpf1 complex, and reduces 25S rRNA, linking 60S assembly to cellular redox status [PMID:41231949]. Loss of MAK16 function causes G1 cell cycle arrest, consistent with the dependence of proliferation on adequate 60S subunit supply [PMID:3045810, PMID:7739558].","teleology":[{"year":1988,"claim":"Established that MAK16 encodes a nuclear protein whose loss blocks cell cycle progression, first linking the gene to proliferation control.","evidence":"Subcellular fractionation of MAK16-LacZ fusions and G1-arrest phenotyping of a temperature-sensitive mak16-1 mutant in yeast","pmids":["3045810"],"confidence":"Medium","gaps":["Molecular basis of the G1 arrest not defined","Nuclear localization shown but sub-nuclear (nucleolar) targeting not yet resolved"]},{"year":1995,"claim":"Connected the cell cycle phenotype to a concrete molecular defect by showing MAK16 is required for the supply of free 60S ribosomal subunits.","evidence":"Sucrose gradient polysome profiling of mak16 mutants with genetic complementation in yeast","pmids":["7739558"],"confidence":"Medium","gaps":["Step in 60S biogenesis affected not identified","Direct molecular role of the protein unknown"]},{"year":2000,"claim":"Refined the localization to the nucleolus and showed this targeting is conserved across species, consistent with a ribosome biogenesis role.","evidence":"GFP-fusion imaging of MAK16 homologs in COS-7 cells plus in vitro CK2 phosphorylation assay","pmids":["10838225"],"confidence":"Medium","gaps":["Functional significance of CK2 phosphorylation untested in vivo","Mechanism of nucleolar function not addressed"]},{"year":2006,"claim":"Pinpointed the rRNA-processing step by showing Mak16 is required for 27SB pre-rRNA stability and downstream 25S/5.8S maturation, and that mere pre-ribosome association is insufficient for function.","evidence":"Northern blot of rRNA intermediates and gradient fractionation in temperature-sensitive mak16-1 yeast","pmids":["16710831"],"confidence":"Medium","gaps":["How Mak16 promotes 27SB stability mechanistically unresolved","Direct partners within the pre-60S particle not defined"]},{"year":2017,"claim":"Provided the structural framework by placing Mak16 in the Nsa1-Rrp1-Rpf1-Mak16 module stabilizing the solvent face of early nucleolar pre-60S intermediates.","evidence":"Cryo-EM of purified early 60S biogenesis intermediates at 3.3-4.5 A from S. cerevisiae","pmids":["29245012"],"confidence":"High","gaps":["Dynamics of module assembly/release not captured","Functional consequence of contacts not perturbed in structures"]},{"year":2022,"claim":"Positioned Mak16 within an ordered maturation pathway by showing eIF4G1 loss causes aberrant retention of Mak16 on pre-60S and impairs PET maturation and 27S processing.","evidence":"Mass spectrometry of purified pre-60S complexes from eIF4G1-deletion yeast with genetic interaction analysis","pmids":["35615984"],"confidence":"Medium","gaps":["Direct vs. indirect role of eIF4G1 in Mak16 release unclear","Mechanism coupling Mak16 retention to PET defects not defined"]},{"year":2024,"claim":"Demonstrated conservation of the module in human cells and its functional coupling to the MTR4 helicase, with mutual interdependence of WDR74-RPF1-MAK16-RRP1 for pre-rRNA cleavage.","evidence":"Co-immunoprecipitation/mass spectrometry and functional knockdown with pre-rRNA processing readout in human cells","pmids":["39706051"],"confidence":"Medium","gaps":["Architecture of the human module relative to yeast not structurally resolved","Direct vs. bridged contacts with MTR4 not mapped"]},{"year":2025,"claim":"Revealed a chemical cofactor basis for Mak16 function: a redox-active [4Fe-4S] cluster required for protein stability and Rpf1 binding, providing a mechanism linking ribosome assembly to redox state.","evidence":"EPR/electrochemistry of reconstituted cluster, in vivo H2O2 treatment, coordination-residue mutagenesis, Mak16-Rpf1 co-IP, and Northern blot for 25S in yeast and human Mak16","pmids":["41231949"],"confidence":"High","gaps":["Physiological redox conditions that toggle the cluster in vivo not defined","Whether cluster loss is reversible/regulated remains open","Structural basis of cluster-dependent Rpf1 interaction not resolved"]},{"year":null,"claim":"How redox-driven Mak16 cluster status is integrated with the broader pre-60S maturation timeline to regulate ribosome output under stress remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No quantitative link between cluster oxidation and cell-cycle/proliferation phenotypes","Upstream Fe/S assembly machinery for Mak16 not identified in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[3,6]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3,4,6]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[4]}],"complexes":["Nsa1-Rrp1-Rpf1-Mak16 pre-60S module","WDR74-RPF1-MAK16-RRP1 module","66S/pre-60S pre-ribosomal particle"],"partners":["RPF1","RRP1","NSA1","WDR74","MTR4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BXY0","full_name":"Protein MAK16 homolog","aliases":["NNP78","Protein RBM13"],"length_aa":300,"mass_kda":35.4,"function":"","subcellular_location":"Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/Q9BXY0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MAK16","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAK16","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MAK16"},"hgnc":{"alias_symbol":["MAK16L"],"prev_symbol":["RBM13"]},"alphafold":{"accession":"Q9BXY0","domains":[{"cath_id":"3.30.390.110","chopping":"5-129","consensus_level":"medium","plddt":93.6491,"start":5,"end":129},{"cath_id":"-","chopping":"161-194","consensus_level":"medium","plddt":75.2809,"start":161,"end":194}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXY0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXY0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXY0-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAK16","jax_strain_url":"https://www.jax.org/strain/search?query=MAK16"},"sequence":{"accession":"Q9BXY0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BXY0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BXY0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXY0"}},"corpus_meta":[{"pmid":"29245012","id":"PMC_29245012","title":"Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes.","date":"2017","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/29245012","citation_count":157,"is_preprint":false},{"pmid":"8552095","id":"PMC_8552095","title":"Paf1p, an RNA polymerase II-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription.","date":"1996","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/8552095","citation_count":122,"is_preprint":false},{"pmid":"7739558","id":"PMC_7739558","title":"Yeast virus propagation depends critically on free 60S ribosomal subunit concentration.","date":"1995","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/7739558","citation_count":94,"is_preprint":false},{"pmid":"6987655","id":"PMC_6987655","title":"\"Superkiller\" mutations suppress chromosomal mutations affecting double-stranded RNA killer plasmid replication in saccharomyces cerevisiae.","date":"1980","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/6987655","citation_count":64,"is_preprint":false},{"pmid":"3332963","id":"PMC_3332963","title":"Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures.","date":"1987","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/3332963","citation_count":57,"is_preprint":false},{"pmid":"15621414","id":"PMC_15621414","title":"Glutathione transferase-like proteins encoded in genomes of yeasts and fungi: insights into evolution of a multifunctional protein superfamily.","date":"2005","source":"FEMS microbiology letters","url":"https://pubmed.ncbi.nlm.nih.gov/15621414","citation_count":57,"is_preprint":false},{"pmid":"387719","id":"PMC_387719","title":"Mak mutants of yeast: mapping and characterization.","date":"1979","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/387719","citation_count":55,"is_preprint":false},{"pmid":"6383953","id":"PMC_6383953","title":"Temperature-sensitive lethal mutations on yeast chromosome I appear to define only a small number of genes.","date":"1984","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/6383953","citation_count":52,"is_preprint":false},{"pmid":"8144453","id":"PMC_8144453","title":"Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the genes in the FUN38-MAK16-SPO7 region.","date":"1994","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/8144453","citation_count":35,"is_preprint":false},{"pmid":"3045810","id":"PMC_3045810","title":"Host function of MAK16: G1 arrest by a mak16 mutant of Saccharomyces cerevisiae.","date":"1988","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/3045810","citation_count":32,"is_preprint":false},{"pmid":"32828126","id":"PMC_32828126","title":"Integrated analysis of RNA-binding proteins in human colorectal cancer.","date":"2020","source":"World journal of surgical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32828126","citation_count":26,"is_preprint":false},{"pmid":"31384924","id":"PMC_31384924","title":"Adaptation to Extreme Environments in an Admixed Human Population from the Atacama Desert.","date":"2019","source":"Genome biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/31384924","citation_count":15,"is_preprint":false},{"pmid":"16710831","id":"PMC_16710831","title":"Mak16p is required for the maturation of 25S and 5.8S rRNAs in the yeast Saccharomyces cerevisiae.","date":"2006","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/16710831","citation_count":13,"is_preprint":false},{"pmid":"10838225","id":"PMC_10838225","title":"SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus.","date":"2000","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/10838225","citation_count":12,"is_preprint":false},{"pmid":"38433915","id":"PMC_38433915","title":"Differential alternative splicing landscape identifies potentially functional RNA binding proteins in early embryonic development in mammals.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/38433915","citation_count":4,"is_preprint":false},{"pmid":"25246723","id":"PMC_25246723","title":"The MAK16 gene of Entamoeba histolytica and its identification in isolates from patients.","date":"2014","source":"The Korean journal of parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/25246723","citation_count":3,"is_preprint":false},{"pmid":"27475537","id":"PMC_27475537","title":"Bacterial versus human sphingosine-1-phosphate lyase (S1PL) in the design of potential S1PL inhibitors.","date":"2016","source":"Bioorganic & medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27475537","citation_count":3,"is_preprint":false},{"pmid":"39706051","id":"PMC_39706051","title":"Pre-ribosomal WDR74 module coordinates the early and late pre-rRNA processing stages for the NVL2-mediated regulation of 60S ribosome biogenesis.","date":"2024","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/39706051","citation_count":3,"is_preprint":false},{"pmid":"35615984","id":"PMC_35615984","title":"Translation initiation factor eIF4G1 modulates assembly of the polypeptide exit tunnel region in yeast ribosome biogenesis.","date":"2022","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/35615984","citation_count":3,"is_preprint":false},{"pmid":"3551912","id":"PMC_3551912","title":"Molecular characterization of chromosomal genes affecting double-stranded RNA replication in Saccharomyces 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macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/42103131","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13659,"output_tokens":2339,"usd":0.038031,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9396,"output_tokens":2942,"usd":0.060265,"stage2_stop_reason":"end_turn"},"total_usd":0.098296,"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\": 1988,\n      \"finding\": \"MAK16 encodes a 306-amino acid nuclear protein (Mr 35,694) with two nuclear localization signal sequences; MAK16-LacZ fusion proteins containing one NLS entered the nucleus as determined by subcellular fractionation, while unfused beta-galactosidase did not. Loss-of-function (mak16-1) causes G1 arrest at non-permissive temperature, with cells remaining mating competent, placing MAK16 function in G1 cell cycle progression.\",\n      \"method\": \"Subcellular fractionation of MAK16-LacZ fusion proteins; cell cycle staging by mating competence assay; DNA sequencing to identify NLS motifs\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct fractionation experiment demonstrating nuclear localization, combined with cell-cycle arrest phenotype from loss-of-function; single lab, two orthogonal methods\",\n      \"pmids\": [\"3045810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"mak16 mutants have decreased free 60S ribosomal subunits, as shown by polysome profile analysis; supplying the normal MAK16 gene restored free 60S subunit levels, L-A copy number, and L-A coat protein to wild-type levels, establishing that MAK16 function is required for 60S ribosomal subunit supply.\",\n      \"method\": \"Sucrose gradient polysome profiling; complementation with single-copy MAK16 gene\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — polysome profiling plus genetic complementation, single lab, two orthogonal methods\",\n      \"pmids\": [\"7739558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The Schistosoma mansoni MAK16 homolog (SmMAK16) is phosphorylated in vitro by human protein kinase CK2. GFP fusions of SmMAK16 localize exclusively to nucleoli (not nuclei) in COS-7 cells, and the yeast and C. elegans homologs similarly direct nucleolar accumulation, establishing nucleolar targeting as a conserved feature of MAK16 family proteins and suggesting a role in 60S subunit biogenesis.\",\n      \"method\": \"In vitro phosphorylation assay with recombinant CK2; transient transfection of GFP fusion constructs in COS-7 cells; fluorescence microscopy\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell imaging of GFP fusions plus in vitro kinase assay; single lab, two orthogonal methods\",\n      \"pmids\": [\"10838225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Mak16p is required for the stability of the 27SB precursor rRNA in S. cerevisiae: at non-permissive temperature, mak16-1 cells show a dramatic decrease in 27SB pre-rRNA levels, leading to loss of mature 25S and 5.8S rRNAs, while 18S and 5S are only moderately affected. The mutant Mak16-1p protein was still associated with the 66S pre-ribosomal complex, indicating that mere association with the 66S pre-RNP is insufficient for function.\",\n      \"method\": \"Northern blot analysis of rRNA processing intermediates in temperature-sensitive mak16-1 yeast; sucrose density gradient fractionation\",\n      \"journal\": \"Yeast (Chichester, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Northern blot rRNA processing combined with gradient fractionation; single lab, two orthogonal methods\",\n      \"pmids\": [\"16710831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structures of early nucleolar 60S biogenesis intermediates at 3.3–4.5 Å resolution reveal that Mak16 functions as part of the Nsa1-Rrp1-Rpf1-Mak16 module, which stabilizes the solvent side of the 60S subunit during early nucleolar pre-60S assembly. The structural snapshots show Mak16 mapped to specific assembly states in the sequential folding pathway.\",\n      \"method\": \"Cryo-electron microscopy (cryo-EM) of purified early 60S biogenesis intermediates from S. cerevisiae; structural modeling at 3.3–4.5 Å resolution\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with functional pathway placement; replicated in independent structural studies\",\n      \"pmids\": [\"29245012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Deletion of eIF4G1 in S. cerevisiae causes abnormal retention of Mak16 (along with Ssf1, Rrp15, Rrp14) on the pre-60S complex, impairing polypeptide exit tunnel (PET) maturation and 27S processing, placing Mak16 in a pathway downstream of eIF4G1 during pre-60S maturation.\",\n      \"method\": \"Mass spectrometry of purified pre-60S complexes from eIF4G1 deletion yeast; genetic interaction analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry of purified complexes combined with genetic deletion; single lab\",\n      \"pmids\": [\"35615984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In human cells, MAK16 functions as part of a pre-ribosomal WDR74 module (comprising WDR74, RPF1, MAK16, and RRP1) identified by co-immunoprecipitation coupled with mass spectrometry. Each component of this module is mutually required for interaction of other members with the RNA helicase MTR4, and all components are required for accurate cleavage of pre-rRNA during 60S biogenesis.\",\n      \"method\": \"Co-immunoprecipitation combined with mass spectrometry; functional knockdown experiments monitoring pre-rRNA processing\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS plus functional knockdown with defined pre-rRNA processing readout; single lab\",\n      \"pmids\": [\"39706051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Mak16 (yeast and human) contains a redox-active [4Fe-4S]2+/1+ cluster with midpoint potential below -500 mV, identified by both in vivo and in vitro methods. Oxidative stress (H2O2) causes [3Fe-4S]1+ cluster formation, destabilizes Mak16, and disrupts its interaction with Rpf1 in vivo. Disruption of Fe/S cluster coordination by mutagenesis destabilized Mak16, impaired Mak16-Rpf1 complex formation, and decreased 25S rRNA levels, establishing the Fe/S cluster as essential for Mak16 function in 60S ribosome biogenesis and as a potential sensor for redox imbalance.\",\n      \"method\": \"In vitro Fe/S cluster characterization (EPR spectroscopy, electrochemistry); in vivo H2O2 treatment; mutagenesis of Fe/S cluster coordination residues; co-immunoprecipitation of Mak16-Rpf1 complex; Northern blot for 25S rRNA levels\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution and characterization of Fe/S cluster combined with mutagenesis, in vivo functional validation, and structural correlation; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"41231949\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAK16 is an essential nucleolar protein that, as part of the Nsa1/Rrp1/Rpf1/Mak16 module, stabilizes the solvent face of early pre-60S ribosomal intermediates and is required for 27SB pre-rRNA stability and maturation of 25S and 5.8S rRNAs; its function critically depends on a redox-active [4Fe-4S] cluster that mediates its interaction with partner Rpf1 and is destabilized by oxidative stress, linking ribosome assembly to cellular redox status.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAK16 is an essential, conserved nucleolar protein required for biogenesis of the large (60S) ribosomal subunit [#1, #2]. Within early nucleolar pre-60S intermediates it acts as a component of the Nsa1-Rrp1-Rpf1-Mak16 module that stabilizes the solvent face of the assembling subunit, with cryo-EM placing it at defined states of the sequential rRNA folding pathway [#4]; an equivalent human pre-ribosomal module (WDR74-RPF1-MAK16-RRP1) couples to the RNA helicase MTR4 and is collectively required for accurate pre-rRNA cleavage during 60S biogenesis [#6]. Functionally, Mak16 is needed for stability of the 27SB pre-rRNA and hence for maturation of mature 25S and 5.8S rRNAs, and its proper release from the pre-60S particle is governed by upstream factors such as eIF4G1 [#3, #5]. Mak16 harbors a redox-active [4Fe-4S]2+/1+ cluster (midpoint potential below -500 mV) that is essential for its stability and for its interaction with the partner Rpf1; oxidative stress converts it to a [3Fe-4S]1+ form, destabilizes the protein, disrupts the Mak16-Rpf1 complex, and reduces 25S rRNA, linking 60S assembly to cellular redox status [#7]. Loss of MAK16 function causes G1 cell cycle arrest, consistent with the dependence of proliferation on adequate 60S subunit supply [#0, #1].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1988,\n      \"claim\": \"Established that MAK16 encodes a nuclear protein whose loss blocks cell cycle progression, first linking the gene to proliferation control.\",\n      \"evidence\": \"Subcellular fractionation of MAK16-LacZ fusions and G1-arrest phenotyping of a temperature-sensitive mak16-1 mutant in yeast\",\n      \"pmids\": [\"3045810\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the G1 arrest not defined\", \"Nuclear localization shown but sub-nuclear (nucleolar) targeting not yet resolved\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Connected the cell cycle phenotype to a concrete molecular defect by showing MAK16 is required for the supply of free 60S ribosomal subunits.\",\n      \"evidence\": \"Sucrose gradient polysome profiling of mak16 mutants with genetic complementation in yeast\",\n      \"pmids\": [\"7739558\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Step in 60S biogenesis affected not identified\", \"Direct molecular role of the protein unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Refined the localization to the nucleolus and showed this targeting is conserved across species, consistent with a ribosome biogenesis role.\",\n      \"evidence\": \"GFP-fusion imaging of MAK16 homologs in COS-7 cells plus in vitro CK2 phosphorylation assay\",\n      \"pmids\": [\"10838225\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional significance of CK2 phosphorylation untested in vivo\", \"Mechanism of nucleolar function not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Pinpointed the rRNA-processing step by showing Mak16 is required for 27SB pre-rRNA stability and downstream 25S/5.8S maturation, and that mere pre-ribosome association is insufficient for function.\",\n      \"evidence\": \"Northern blot of rRNA intermediates and gradient fractionation in temperature-sensitive mak16-1 yeast\",\n      \"pmids\": [\"16710831\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How Mak16 promotes 27SB stability mechanistically unresolved\", \"Direct partners within the pre-60S particle not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the structural framework by placing Mak16 in the Nsa1-Rrp1-Rpf1-Mak16 module stabilizing the solvent face of early nucleolar pre-60S intermediates.\",\n      \"evidence\": \"Cryo-EM of purified early 60S biogenesis intermediates at 3.3-4.5 A from S. cerevisiae\",\n      \"pmids\": [\"29245012\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of module assembly/release not captured\", \"Functional consequence of contacts not perturbed in structures\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Positioned Mak16 within an ordered maturation pathway by showing eIF4G1 loss causes aberrant retention of Mak16 on pre-60S and impairs PET maturation and 27S processing.\",\n      \"evidence\": \"Mass spectrometry of purified pre-60S complexes from eIF4G1-deletion yeast with genetic interaction analysis\",\n      \"pmids\": [\"35615984\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect role of eIF4G1 in Mak16 release unclear\", \"Mechanism coupling Mak16 retention to PET defects not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated conservation of the module in human cells and its functional coupling to the MTR4 helicase, with mutual interdependence of WDR74-RPF1-MAK16-RRP1 for pre-rRNA cleavage.\",\n      \"evidence\": \"Co-immunoprecipitation/mass spectrometry and functional knockdown with pre-rRNA processing readout in human cells\",\n      \"pmids\": [\"39706051\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Architecture of the human module relative to yeast not structurally resolved\", \"Direct vs. bridged contacts with MTR4 not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a chemical cofactor basis for Mak16 function: a redox-active [4Fe-4S] cluster required for protein stability and Rpf1 binding, providing a mechanism linking ribosome assembly to redox state.\",\n      \"evidence\": \"EPR/electrochemistry of reconstituted cluster, in vivo H2O2 treatment, coordination-residue mutagenesis, Mak16-Rpf1 co-IP, and Northern blot for 25S in yeast and human Mak16\",\n      \"pmids\": [\"41231949\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological redox conditions that toggle the cluster in vivo not defined\", \"Whether cluster loss is reversible/regulated remains open\", \"Structural basis of cluster-dependent Rpf1 interaction not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How redox-driven Mak16 cluster status is integrated with the broader pre-60S maturation timeline to regulate ribosome output under stress remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No quantitative link between cluster oxidation and cell-cycle/proliferation phenotypes\", \"Upstream Fe/S assembly machinery for Mak16 not identified in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3, 4, 6]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\n      \"Nsa1-Rrp1-Rpf1-Mak16 pre-60S module\",\n      \"WDR74-RPF1-MAK16-RRP1 module\",\n      \"66S/pre-60S pre-ribosomal particle\"\n    ],\n    \"partners\": [\n      \"RPF1\",\n      \"RRP1\",\n      \"NSA1\",\n      \"WDR74\",\n      \"MTR4\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}