{"gene":"RPL8","run_date":"2026-06-10T07:46:26","timeline":{"discoveries":[{"year":2015,"finding":"Human ribosomal protein uL2 (RPL8) contains a eukaryote-specific hydroxylated His216 in its unstructured C-terminal loop that contacts 28S rRNA helix H93 near the peptidyl transferase center; the hydroxyl group stabilizes a protein conformation favorable for H93 binding, and this binding induces structural rearrangement in regions close to the peptidyl transferase center (affecting universally conserved nucleotides U4532/C4447).","method":"Chemical probing with hydroxyl radicals and other probes on RNA mimicking 28S rRNA domain V segment, comparing natural (hydroxylated) vs. recombinant (unmodified) uL2 binding","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical binding assay with two protein forms and multiple chemical probes, single lab","pmids":["25702831"],"is_preprint":false},{"year":2015,"finding":"NO66, a JmjC domain-containing protein, acts as a hydroxylase for ribosomal protein Rpl8 (RPL8/uL2); NO66 oligomerization (tetramerization) is required for efficient catalysis, and it recognizes a consensus sequence motif NHXH on Rpl8 (residues 204–224); structural shifts in NO66 subunit positions occur upon substrate binding.","method":"Crystal structure of NO66(176-C) complexed with Rpl8(204-224) peptide; in vitro hydroxylase activity assay; mutagenesis of NO66; oligomerization analysis","journal":"Acta crystallographica. Section D, Biological crystallography","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with biochemical validation and mutagenesis in same study","pmids":["26327385"],"is_preprint":false},{"year":2022,"finding":"Missense variants in RPL8 (encoding uL2, a protein of the 60S large ribosomal subunit) found in Diamond-Blackfan anemia patients produce functionally deficient proteins that impair ribosome production, establishing RPL8 as a DBA-associated gene.","method":"Functional studies in patient-derived lymphoblastoid cells and yeast models assessing ribosome biogenesis/production","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function cellular and yeast model with defined ribosome production phenotype, single lab, two orthogonal model systems","pmids":["34961992"],"is_preprint":false},{"year":2025,"finding":"During bacterial 50S ribosomal subunit assembly, the GTPase YsxC acts as a placeholder for ribosomal protein uL2 (RPL8 ortholog): YsxC occupies the uL2 binding site in the 44.5S assembly intermediate, controlling timing of rRNA helix folding; once YsxC is released, uL2 binds a 'primordial' site and the remaining helices fold to mature conformation.","method":"Cryo-EM structure of Bacillus subtilis 44.5S assembly intermediate with YsxC; biochemical assembly assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with biochemical validation establishing placeholder mechanism","pmids":["41148149"],"is_preprint":false},{"year":2022,"finding":"RPL8 silencing in hepatocellular carcinoma cells inhibits cell proliferation, migration, invasion, and glycolysis, and these effects are mediated through the mTORC1 signaling pathway; the upstream transcription factor USF1 regulates RPL8 expression, and USF1 overexpression rescues the inhibitory effects of RPL8 silencing.","method":"siRNA knockdown of RPL8, cell viability/proliferation/migration/invasion assays, glycolysis measurement, bioinformatic pathway analysis, USF1 overexpression rescue experiment","journal":"Human cell","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway placement primarily from bioinformatics with partial rescue experiment, no direct biochemical evidence for mTORC1 interaction","pmids":["36577883"],"is_preprint":false},{"year":2024,"finding":"RPL8 knockdown in hepatocellular carcinoma cells triggers ferroptosis by increasing lipid peroxidation (elevated 4-HNE levels) and altering amino acid metabolism (glycine, glutamate, cysteine), upregulating glutathione synthase (GSS) protein and enhancing GSH synthesis; tumor growth was suppressed in xenograft models upon RPL8 silencing.","method":"siRNA knockdown of RPL8 in HCC cell lines; xenograft tumor growth assay; 4-HNE measurement; GSH pathway protein analysis; ferroptosis inhibitor (Fer-1) rescue","journal":"Archives of medical science : AMS","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, functional knockdown with ferroptosis readout but no direct molecular mechanism for GSH regulation established","pmids":["41078929"],"is_preprint":false}],"current_model":"RPL8 (uL2) is a protein of the 60S large ribosomal subunit whose C-terminal loop contacts 28S rRNA helix H93 near the peptidyl transferase center; a eukaryote-specific hydroxylation of His216 (installed by the JmjC-domain hydroxylase NO66, which requires oligomerization for catalytic efficiency) stabilizes the loop conformation and induces structural rearrangements near the PTC; in bacteria, the GTPase YsxC acts as a placeholder for the uL2 binding site during 50S assembly, controlling the timing of rRNA helix folding before uL2 itself occupies the mature site; loss-of-function RPL8 variants impair ribosome production and cause Diamond-Blackfan anemia."},"narrative":{"mechanistic_narrative":"RPL8 (uL2) is a structural protein of the 60S large ribosomal subunit essential for ribosome production, and human loss-of-function missense variants impair ribosome biogenesis and cause Diamond-Blackfan anemia [PMID:34961992]. Its function centers on an unstructured C-terminal loop that contacts 28S rRNA helix H93 near the peptidyl transferase center; a eukaryote-specific hydroxylation of His216 within this loop stabilizes a binding-competent conformation, and uL2 engagement of H93 induces structural rearrangements at universally conserved nucleotides adjacent to the PTC [PMID:25702831]. The His216 hydroxylation is installed by the JmjC-domain hydroxylase NO66, which recognizes an NHXH motif in the uL2 residue 204–224 region and requires oligomerization for efficient catalysis [PMID:26327385]. The placement of uL2 during large-subunit assembly is tightly ordered: in bacteria the GTPase YsxC occupies the uL2 binding site in an assembly intermediate as a placeholder, controlling the timing of rRNA helix folding before uL2 itself binds and the surrounding helices mature [PMID:41148149].","teleology":[{"year":2015,"claim":"Established how uL2 contributes to peptidyl transferase center architecture and why a eukaryote-specific His216 modification matters, by showing the hydroxyl stabilizes the C-terminal loop conformation for H93 binding.","evidence":"Hydroxyl radical and chemical probing of an rRNA mimic comparing natural hydroxylated vs. recombinant unmodified uL2","pmids":["25702831"],"confidence":"Medium","gaps":["Done on an rRNA segment mimic rather than intact 28S rRNA/assembled ribosome","Functional consequence of His216 hydroxylation for translation not measured","Single lab, in vitro binding readout"]},{"year":2015,"claim":"Identified the enzyme and recognition logic for uL2 modification, defining NO66 as the His216 hydroxylase and the structural requirements for its catalysis.","evidence":"Crystal structure of NO66 bound to an Rpl8(204-224) peptide with in vitro hydroxylase assays, mutagenesis, and oligomerization analysis","pmids":["26327385"],"confidence":"High","gaps":["Does not establish the cellular consequence of losing the modification","Whether hydroxylation is co- or post-assembly is unresolved"]},{"year":2022,"claim":"Connected RPL8 directly to human disease, showing that deficient variants impair ribosome production and cause Diamond-Blackfan anemia.","evidence":"Functional assays of patient missense variants in patient-derived lymphoblastoid cells and yeast models","pmids":["34961992"],"confidence":"Medium","gaps":["Specific step of ribosome biogenesis disrupted not pinpointed","Genotype-phenotype relationships across variants not detailed"]},{"year":2025,"claim":"Resolved the timing and ordering of uL2 incorporation during large-subunit assembly, showing a GTPase placeholder controls rRNA folding before uL2 binds.","evidence":"Cryo-EM of a Bacillus subtilis 44.5S assembly intermediate bound to YsxC with biochemical assembly assays","pmids":["41148149"],"confidence":"High","gaps":["Established in bacteria; conservation of an equivalent placeholder in eukaryotic 60S assembly not shown","Trigger for YsxC release and handoff to uL2 not defined"]},{"year":null,"claim":"Whether the reported pro-tumorigenic roles of RPL8 in hepatocellular carcinoma reflect a direct extra-ribosomal mechanism or downstream consequences of altered ribosome output remains unresolved.","evidence":"Open question","pmids":[],"confidence":"Low","gaps":["mTORC1 pathway placement rests on bioinformatics with only partial USF1 rescue, no direct biochemical interaction","GSH/ferroptosis regulation lacks a defined molecular mechanism","No link drawn between cancer phenotypes and the ribosome-assembly/PTC functions of uL2"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[0,2,3]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[3]}],"complexes":["60S large ribosomal subunit"],"partners":["NO66","YSXC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P62917","full_name":"Large ribosomal subunit protein uL2","aliases":["60S ribosomal protein L8"],"length_aa":257,"mass_kda":28.0,"function":"Component of the large ribosomal subunit. The ribosome is a large ribonucleoprotein complex responsible for the synthesis of proteins in the cell","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P62917/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RPL8","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":[{"gene":"EIF2S3","stoichiometry":10.0},{"gene":"ENY2","stoichiometry":10.0},{"gene":"RACK1","stoichiometry":10.0},{"gene":"RBM8A","stoichiometry":10.0},{"gene":"RPL11","stoichiometry":10.0},{"gene":"RPL4","stoichiometry":10.0},{"gene":"RPL5","stoichiometry":10.0},{"gene":"RPS16","stoichiometry":10.0},{"gene":"SRP19","stoichiometry":10.0},{"gene":"SRP68","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/search/RPL8","total_profiled":1310},"omim":[{"mim_id":"604177","title":"RIBOSOMAL PROTEIN L8; RPL8","url":"https://www.omim.org/entry/604177"},{"mim_id":"105650","title":"DIAMOND-BLACKFAN ANEMIA 1; DBA1","url":"https://www.omim.org/entry/105650"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"},{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RPL8"},"hgnc":{"alias_symbol":["L8","uL2"],"prev_symbol":[]},"alphafold":{"accession":"P62917","domains":[{"cath_id":"2.30.30.30","chopping":"33-165","consensus_level":"medium","plddt":96.8636,"start":33,"end":165}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P62917","model_url":"https://alphafold.ebi.ac.uk/files/AF-P62917-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P62917-F1-predicted_aligned_error_v6.png","plddt_mean":95.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RPL8","jax_strain_url":"https://www.jax.org/strain/search?query=RPL8"},"sequence":{"accession":"P62917","fasta_url":"https://rest.uniprot.org/uniprotkb/P62917.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P62917/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P62917"}},"corpus_meta":[{"pmid":"2567340","id":"PMC_2567340","title":"Gene UL2 of herpes simplex virus type 1 encodes a uracil-DNA glycosylase.","date":"1989","source":"The Journal of general virology","url":"https://pubmed.ncbi.nlm.nih.gov/2567340","citation_count":84,"is_preprint":false},{"pmid":"8396663","id":"PMC_8396663","title":"A 3' coterminal gene cluster in pseudorabies virus contains herpes simplex virus UL1, UL2, and UL3 gene homologs and a unique UL3.5 open reading frame.","date":"1993","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/8396663","citation_count":49,"is_preprint":false},{"pmid":"7483276","id":"PMC_7483276","title":"Identification and transcriptional analysis of a 3'-coterminal gene cluster containing UL1, UL2, UL3, and UL3.5 open reading frames of bovine herpesvirus-1.","date":"1995","source":"Virology","url":"https://pubmed.ncbi.nlm.nih.gov/7483276","citation_count":35,"is_preprint":false},{"pmid":"32477319","id":"PMC_32477319","title":"Herpes Simplex Virus 1 UL2 Inhibits the TNF-α-Mediated NF-κB Activity by Interacting With p65/p50.","date":"2020","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32477319","citation_count":24,"is_preprint":false},{"pmid":"1343779","id":"PMC_1343779","title":"Sequence analysis of a mosquito ribosomal protein rpL8 gene and its upstream regulatory region.","date":"1992","source":"Insect molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/1343779","citation_count":22,"is_preprint":false},{"pmid":"25702831","id":"PMC_25702831","title":"Hydroxylated histidine of human ribosomal protein uL2 is involved in maintaining the local structure of 28S rRNA in the ribosomal peptidyl transferase center.","date":"2015","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/25702831","citation_count":20,"is_preprint":false},{"pmid":"28667419","id":"PMC_28667419","title":"Regulation of DNA methylation on EEF1D and RPL8 expression in cattle.","date":"2017","source":"Genetica","url":"https://pubmed.ncbi.nlm.nih.gov/28667419","citation_count":16,"is_preprint":false},{"pmid":"36577883","id":"PMC_36577883","title":"Silencing RPL8 inhibits the progression of hepatocellular carcinoma by down-regulating the mTORC1 signalling pathway.","date":"2022","source":"Human cell","url":"https://pubmed.ncbi.nlm.nih.gov/36577883","citation_count":15,"is_preprint":false},{"pmid":"27865090","id":"PMC_27865090","title":"Characterization of the subcellular localization and nuclear import molecular mechanisms of herpes simplex virus 1 UL2.","date":"2017","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27865090","citation_count":14,"is_preprint":false},{"pmid":"25893343","id":"PMC_25893343","title":"Dysregulated COL3A1 and RPL8, RPS16, and RPS23 in Disc Degeneration Revealed by Bioinformatics Methods.","date":"2015","source":"Spine","url":"https://pubmed.ncbi.nlm.nih.gov/25893343","citation_count":13,"is_preprint":false},{"pmid":"32035424","id":"PMC_32035424","title":"The nuclear localization signal-mediated nuclear targeting of herpes simplex virus 1 early protein UL2 is important for efficient viral production.","date":"2020","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/32035424","citation_count":12,"is_preprint":false},{"pmid":"31520776","id":"PMC_31520776","title":"A novel mutation in the promoter region of RPL8 regulates milk fat traits in dairy cattle by binding transcription factor Pax6.","date":"2019","source":"Biochimica et biophysica acta. Molecular and cell biology of lipids","url":"https://pubmed.ncbi.nlm.nih.gov/31520776","citation_count":11,"is_preprint":false},{"pmid":"8383240","id":"PMC_8383240","title":"Transcriptional and translational analyses of the UL2 gene of equine herpesvirus 1: a homolog of UL55 of herpes simplex virus type 1 that is maintained in the genome of defective interfering particles.","date":"1993","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/8383240","citation_count":10,"is_preprint":false},{"pmid":"26327385","id":"PMC_26327385","title":"Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8.","date":"2015","source":"Acta crystallographica. 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W.V.P.A","url":"https://pubmed.ncbi.nlm.nih.gov/28609137","citation_count":3,"is_preprint":false},{"pmid":"33002000","id":"PMC_33002000","title":"The ex planta signal activity of a Medicago ribosomal uL2 protein suggests a moonlighting role in controlling secondary rhizobial infection.","date":"2020","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/33002000","citation_count":3,"is_preprint":false},{"pmid":"9013493","id":"PMC_9013493","title":"The UL2 open reading frame of bovine herpesvirus 1 encodes a uracil-DNA glycosylase.","date":"1996","source":"Microbiology and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/9013493","citation_count":2,"is_preprint":false},{"pmid":"40801158","id":"PMC_40801158","title":"Pathogenic mechanisms and molecular features of a novel UL2 gene-deficient duck enteritis virus endemic to China.","date":"2025","source":"Virulence","url":"https://pubmed.ncbi.nlm.nih.gov/40801158","citation_count":1,"is_preprint":false},{"pmid":"41148149","id":"PMC_41148149","title":"YsxC is a placeholder for ribosomal protein uL2 during 50S ribosomal subunit assembly.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/41148149","citation_count":1,"is_preprint":false},{"pmid":"41078929","id":"PMC_41078929","title":"Uncovering the role of RPL8 in glutathione synthesis-dependent ferroptosis control in hepatocellular carcinoma.","date":"2024","source":"Archives of medical science : AMS","url":"https://pubmed.ncbi.nlm.nih.gov/41078929","citation_count":0,"is_preprint":false},{"pmid":"42249740","id":"PMC_42249740","title":"Dissecting Immune Cell Ferroptosis via Single-Cell Multi-Omics Identifies RPL8 as a Potential Therapeutic Target for Depression.","date":"2026","source":"FASEB journal : official publication of the Federation of American Societies for 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calcarata</i>","date":"2025-07-31","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.28.667322","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15067,"output_tokens":1679,"usd":0.035193,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8500,"output_tokens":2004,"usd":0.0463,"stage2_stop_reason":"end_turn"},"total_usd":0.081493,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"Human ribosomal protein uL2 (RPL8) contains a eukaryote-specific hydroxylated His216 in its unstructured C-terminal loop that contacts 28S rRNA helix H93 near the peptidyl transferase center; the hydroxyl group stabilizes a protein conformation favorable for H93 binding, and this binding induces structural rearrangement in regions close to the peptidyl transferase center (affecting universally conserved nucleotides U4532/C4447).\",\n      \"method\": \"Chemical probing with hydroxyl radicals and other probes on RNA mimicking 28S rRNA domain V segment, comparing natural (hydroxylated) vs. recombinant (unmodified) uL2 binding\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical binding assay with two protein forms and multiple chemical probes, single lab\",\n      \"pmids\": [\"25702831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NO66, a JmjC domain-containing protein, acts as a hydroxylase for ribosomal protein Rpl8 (RPL8/uL2); NO66 oligomerization (tetramerization) is required for efficient catalysis, and it recognizes a consensus sequence motif NHXH on Rpl8 (residues 204–224); structural shifts in NO66 subunit positions occur upon substrate binding.\",\n      \"method\": \"Crystal structure of NO66(176-C) complexed with Rpl8(204-224) peptide; in vitro hydroxylase activity assay; mutagenesis of NO66; oligomerization analysis\",\n      \"journal\": \"Acta crystallographica. Section D, Biological crystallography\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with biochemical validation and mutagenesis in same study\",\n      \"pmids\": [\"26327385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Missense variants in RPL8 (encoding uL2, a protein of the 60S large ribosomal subunit) found in Diamond-Blackfan anemia patients produce functionally deficient proteins that impair ribosome production, establishing RPL8 as a DBA-associated gene.\",\n      \"method\": \"Functional studies in patient-derived lymphoblastoid cells and yeast models assessing ribosome biogenesis/production\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function cellular and yeast model with defined ribosome production phenotype, single lab, two orthogonal model systems\",\n      \"pmids\": [\"34961992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"During bacterial 50S ribosomal subunit assembly, the GTPase YsxC acts as a placeholder for ribosomal protein uL2 (RPL8 ortholog): YsxC occupies the uL2 binding site in the 44.5S assembly intermediate, controlling timing of rRNA helix folding; once YsxC is released, uL2 binds a 'primordial' site and the remaining helices fold to mature conformation.\",\n      \"method\": \"Cryo-EM structure of Bacillus subtilis 44.5S assembly intermediate with YsxC; biochemical assembly assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with biochemical validation establishing placeholder mechanism\",\n      \"pmids\": [\"41148149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RPL8 silencing in hepatocellular carcinoma cells inhibits cell proliferation, migration, invasion, and glycolysis, and these effects are mediated through the mTORC1 signaling pathway; the upstream transcription factor USF1 regulates RPL8 expression, and USF1 overexpression rescues the inhibitory effects of RPL8 silencing.\",\n      \"method\": \"siRNA knockdown of RPL8, cell viability/proliferation/migration/invasion assays, glycolysis measurement, bioinformatic pathway analysis, USF1 overexpression rescue experiment\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway placement primarily from bioinformatics with partial rescue experiment, no direct biochemical evidence for mTORC1 interaction\",\n      \"pmids\": [\"36577883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RPL8 knockdown in hepatocellular carcinoma cells triggers ferroptosis by increasing lipid peroxidation (elevated 4-HNE levels) and altering amino acid metabolism (glycine, glutamate, cysteine), upregulating glutathione synthase (GSS) protein and enhancing GSH synthesis; tumor growth was suppressed in xenograft models upon RPL8 silencing.\",\n      \"method\": \"siRNA knockdown of RPL8 in HCC cell lines; xenograft tumor growth assay; 4-HNE measurement; GSH pathway protein analysis; ferroptosis inhibitor (Fer-1) rescue\",\n      \"journal\": \"Archives of medical science : AMS\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, functional knockdown with ferroptosis readout but no direct molecular mechanism for GSH regulation established\",\n      \"pmids\": [\"41078929\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RPL8 (uL2) is a protein of the 60S large ribosomal subunit whose C-terminal loop contacts 28S rRNA helix H93 near the peptidyl transferase center; a eukaryote-specific hydroxylation of His216 (installed by the JmjC-domain hydroxylase NO66, which requires oligomerization for catalytic efficiency) stabilizes the loop conformation and induces structural rearrangements near the PTC; in bacteria, the GTPase YsxC acts as a placeholder for the uL2 binding site during 50S assembly, controlling the timing of rRNA helix folding before uL2 itself occupies the mature site; loss-of-function RPL8 variants impair ribosome production and cause Diamond-Blackfan anemia.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RPL8 (uL2) is a structural protein of the 60S large ribosomal subunit essential for ribosome production, and human loss-of-function missense variants impair ribosome biogenesis and cause Diamond-Blackfan anemia [#2]. Its function centers on an unstructured C-terminal loop that contacts 28S rRNA helix H93 near the peptidyl transferase center; a eukaryote-specific hydroxylation of His216 within this loop stabilizes a binding-competent conformation, and uL2 engagement of H93 induces structural rearrangements at universally conserved nucleotides adjacent to the PTC [#0]. The His216 hydroxylation is installed by the JmjC-domain hydroxylase NO66, which recognizes an NHXH motif in the uL2 residue 204\\u2013224 region and requires oligomerization for efficient catalysis [#1]. The placement of uL2 during large-subunit assembly is tightly ordered: in bacteria the GTPase YsxC occupies the uL2 binding site in an assembly intermediate as a placeholder, controlling the timing of rRNA helix folding before uL2 itself binds and the surrounding helices mature [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Established how uL2 contributes to peptidyl transferase center architecture and why a eukaryote-specific His216 modification matters, by showing the hydroxyl stabilizes the C-terminal loop conformation for H93 binding.\",\n      \"evidence\": \"Hydroxyl radical and chemical probing of an rRNA mimic comparing natural hydroxylated vs. recombinant unmodified uL2\",\n      \"pmids\": [\n        \"25702831\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Done on an rRNA segment mimic rather than intact 28S rRNA/assembled ribosome\",\n        \"Functional consequence of His216 hydroxylation for translation not measured\",\n        \"Single lab, in vitro binding readout\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the enzyme and recognition logic for uL2 modification, defining NO66 as the His216 hydroxylase and the structural requirements for its catalysis.\",\n      \"evidence\": \"Crystal structure of NO66 bound to an Rpl8(204-224) peptide with in vitro hydroxylase assays, mutagenesis, and oligomerization analysis\",\n      \"pmids\": [\n        \"26327385\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Does not establish the cellular consequence of losing the modification\",\n        \"Whether hydroxylation is co- or post-assembly is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected RPL8 directly to human disease, showing that deficient variants impair ribosome production and cause Diamond-Blackfan anemia.\",\n      \"evidence\": \"Functional assays of patient missense variants in patient-derived lymphoblastoid cells and yeast models\",\n      \"pmids\": [\n        \"34961992\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific step of ribosome biogenesis disrupted not pinpointed\",\n        \"Genotype-phenotype relationships across variants not detailed\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the timing and ordering of uL2 incorporation during large-subunit assembly, showing a GTPase placeholder controls rRNA folding before uL2 binds.\",\n      \"evidence\": \"Cryo-EM of a Bacillus subtilis 44.5S assembly intermediate bound to YsxC with biochemical assembly assays\",\n      \"pmids\": [\n        \"41148149\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Established in bacteria; conservation of an equivalent placeholder in eukaryotic 60S assembly not shown\",\n        \"Trigger for YsxC release and handoff to uL2 not defined\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether the reported pro-tumorigenic roles of RPL8 in hepatocellular carcinoma reflect a direct extra-ribosomal mechanism or downstream consequences of altered ribosome output remains unresolved.\",\n      \"evidence\": \"Open question\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"mTORC1 pathway placement rests on bioinformatics with only partial USF1 rescue, no direct biochemical interaction\",\n        \"GSH/ferroptosis regulation lacks a defined molecular mechanism\",\n        \"No link drawn between cancer phenotypes and the ribosome-assembly/PTC functions of uL2\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\n        \"term_id\": \"GO:0005198\",\n        \"supporting_discovery_ids\": [\n          0,\n          2,\n          3\n        ]\n      },\n      {\n        \"term_id\": \"GO:0003723\",\n        \"supporting_discovery_ids\": [\n          0\n        ]\n      }\n    ],\n    \"localization\": [\n      {\n        \"term_id\": \"GO:0005840\",\n        \"supporting_discovery_ids\": [\n          0,\n          2,\n          3\n        ]\n      }\n    ],\n    \"pathway\": [\n      {\n        \"term_id\": \"R-HSA-8953854\",\n        \"supporting_discovery_ids\": [\n          2,\n          3\n        ]\n      },\n      {\n        \"term_id\": \"R-HSA-1852241\",\n        \"supporting_discovery_ids\": [\n          3\n        ]\n      }\n    ],\n    \"complexes\": [\n      \"60S large ribosomal subunit\"\n    ],\n    \"partners\": [\n      \"NO66\",\n      \"YsxC\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}