{"gene":"RPL36A","run_date":"2026-06-10T07:46:26","timeline":{"discoveries":[{"year":2004,"finding":"Over-expressed L36a/RPL36A ribosomal protein localizes to the nucleoli of cells, and this localization is controlled by the N-terminal or the internal tetrapeptide consensus sequence with its adjacent N-terminal domain.","method":"Ectopic over-expression with subcellular localization imaging (nucleolar localization assay); N-terminal domain mapping","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment with domain mapping in single lab, but no functional reconstitution or structural validation","pmids":["14752831"],"is_preprint":false},{"year":2004,"finding":"Over-expression of RPL36A leads to enhanced colony formation and cell proliferation via accelerated cell cycling; antisense cDNA effectively reversed these effects, establishing a direct role for RPL36A in promoting cellular proliferation.","method":"Ectopic over-expression and antisense knockdown with colony formation assay, proliferation assay, and cell cycle analysis","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function with defined cellular phenotypes, single lab","pmids":["14752831"],"is_preprint":false},{"year":2009,"finding":"The large subunit ribosomal protein RPL36A-like (RPL36AL/eL42) contacts the CCA end of P-site bound tRNA in human 80S ribosomes, placing it at or near the binding site for the tRNA substrate of the peptidyl transferase reaction.","method":"Periodate-oxidized tRNA (zero-length affinity labeling reagent) cross-linking to human 80S ribosomes, with competition by intact tRNA and protein identification by mass spectrometry","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro affinity labeling with direct chemical cross-linking and MS identification, competition assay, single lab","pmids":["19647033"],"is_preprint":false},{"year":1988,"finding":"Rat ribosomal protein L36a (homolog of human RPL36A) contains 105 amino acids (N-terminal Met removed post-translationally) and is homologous to human HL44 and yeast protein 44, suggesting conservation of the L44e family at the peptidyl transferase center.","method":"cDNA sequencing of recombinant clone, N-terminal amino acid sequencing, Southern hybridization for gene copy number","journal":"DNA (Mary Ann Liebert, Inc.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein sequencing confirmed by cDNA sequence, single lab; N-terminal methionine removal established as PTM","pmids":["3396452"],"is_preprint":false},{"year":2017,"finding":"The monomethylated Gln-51 and Lys-53 residues within the 47GFGGQTK53 motif of human eL42 (RPL36A) mediate interaction with the methylated GGQ motif of eRF1 via hydrophobic contacts between methyl groups; eL42 also interacts with tRNA (nanomolar KD) and 28S rRNA (picomolar KD) through strong binding affinities.","method":"Biacore surface plasmon resonance binding assay using recombinant wild-type and mutant eL42 proteins (GGQTK deletion, Q51E, K53Q, Q51A/K53A double mutant) against eRF1, tRNA, and 28S rRNA","journal":"The open biochemistry journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro binding assay with site-directed mutagenesis, single lab, multiple mutants tested","pmids":["28567122"],"is_preprint":false},{"year":2018,"finding":"The GGQ minidomain and neighboring region of eL42 (RPL36A ortholog) is critical for ribosomal function in S. pombe; mutations within or adjacent to the GGQ minidomain fail to complement wild-type eL42 and cause growth defects, reduced poly(Phe) synthesis, and reduced peptidyl transferase activity toward puromycin. A pKa of 6.95 was measured for the side chain of Lys-55/Arg-55, substantially less than a free Lys or Arg, indicating involvement in catalysis.","method":"Site-directed mutagenesis, genetic complementation in S. pombe, in vitro poly(Phe) synthesis assay, peptidyl transferase (puromycin) assay, pKa measurement","journal":"Biochimie","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal in vitro and in vivo methods (mutagenesis, genetic complementation, biochemical assays), rigorous mechanistic dissection of catalytic contribution","pmids":["30550856"],"is_preprint":false},{"year":2019,"finding":"The lysine methyltransferase SET7/9 interacts with eL42 (RPL36A) via its N-terminal MORN domain, and methylates eL42 at three lysines (Lys53, Lys80, Lys100); SET7/9-mediated methylation of eL42 affects global protein translation.","method":"Yeast two-hybrid screening, co-immunoprecipitation, GST pulldown, site-directed mutagenesis of methylation sites, puromycin incorporation assay for translation","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation with mutagenesis, reciprocal co-IP and GST pulldown, functional translation assay, single lab with multiple orthogonal methods","pmids":["31751593"],"is_preprint":false},{"year":2016,"finding":"Elevated levels of eL42 (RPL36A) modulate Hsp90α expression under both normal and heat shock conditions in rhabdomyosarcoma cells; polysome profiling showed selective translation of Hsp90α mRNA while global translation is inhibited during heat stress.","method":"Polysome profiling, manipulation of eL42 levels in rhabdomyosarcoma cells with measurement of Hsp90α expression and sensitivity to Hsp90 inhibitor 17-AAG","journal":"Translation (Austin, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — polysome profiling with functional drug sensitivity assay, single lab","pmids":["28090422"],"is_preprint":false},{"year":2017,"finding":"RPL36A and DOM34 influence CAP-independent translation in yeast and affect expression of HSP82 and HSC82 heat shock proteins in response to acetic acid stress.","method":"Gene deletion/genetic analysis in S. cerevisiae, monitoring of HSP protein expression and CAP-independent translation under acetic acid and heat stress","journal":"PeerJ","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic observations, single lab, limited mechanistic resolution on pathway position","pmids":["29158977"],"is_preprint":false},{"year":2021,"finding":"Knockdown of RPL36A in oral squamous cell carcinoma cells increased radiosensitivity by sensitizing cells to DNA damage, promoting G2/M cell cycle arrest, and augmenting irradiation-induced apoptosis.","method":"siRNA knockdown of RPL36A in OSCC cell lines, irradiation, measurement of DNA damage markers, cell cycle analysis by flow cytometry, apoptosis assay","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotypes (DNA damage, cell cycle, apoptosis), single lab","pmids":["34830778"],"is_preprint":false},{"year":2023,"finding":"Cooperative interactions involving eL42 (RPL36A) residues Q45, Q51, and K53 within the conserved QSGYGGQTK motif are critical for the human ribosome elongation cycle specifically at the step of deacylated tRNA dissociation from the E site; double substitutions at positions 45+51 or 45+53 decrease polysomes without affecting 60S or 80S assembly.","method":"Site-directed mutagenesis of conserved motif residues in FLAG-tagged eL42, expression in HEK293T cells, polysome profiling with Western blotting, tRNA binding assay, peptidyl transferase activity assay","journal":"Biochimie","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with multiple orthogonal assays (polysome profiling, tRNA binding, enzymatic activity) in human cells, single lab","pmids":["37716853"],"is_preprint":false},{"year":2024,"finding":"RPL36A depletion in colorectal cancer cells diminishes phosphorylated ERK levels and subsequently reduces expression of c-Myc and ELK1; the tumor-suppressive effects of RPL36A knockdown are negated by an ERK activator, placing RPL36A upstream of the MAPK/ERK pathway.","method":"siRNA knockdown of RPL36A in CRC cell lines, Western blotting for pERK/c-Myc/ELK1, rescue with ERK activator, tumor xenograft growth assay","journal":"Translational oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via rescue experiment with ERK activator, loss-of-function with defined signaling phenotype, single lab","pmids":["39489085"],"is_preprint":false},{"year":2022,"finding":"In S. pombe, the level of Rpl42p (eL42/RPL36A ortholog) in actively translating ribosomes varies depending on which 40S ribosomal protein paralog is present, identifying variation in eL42 incorporation as a potential form of ribosome heterogeneity; phenotypic differences of rps8 paralog deletions reside in Rpl42p level variation rather than in protein sequence differences between the Rps8 paralogs.","method":"Genetic deletion of paralog genes in fission yeast, polysome profiling, Western blotting to quantify Rpl42p in translating fractions, epistasis analysis","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus polysome fractionation and Western blotting, single lab","pmids":["35954225"],"is_preprint":false}],"current_model":"RPL36A (eL42) is a component of the large (60S) ribosomal subunit whose conserved GGQ-containing motif (47GFGGQTK53) directly contacts the CCA end of P-site tRNA, contributes to peptidyl transferase activity and the elongation cycle (specifically deacylated tRNA release from the E site), is post-translationally methylated by SET7/9 at Lys53/Lys80/Lys100 (which affects global translation), interacts with eRF1 via methylated GGQ contacts during termination, is subject to N-terminal methionine removal, localizes to nucleoli via its N-terminal domain, and extra-ribosomally activates the MAPK/ERK signaling pathway to promote proliferation in cancer contexts."},"narrative":{"mechanistic_narrative":"RPL36A (eL42) is a component of the large (60S) ribosomal subunit that contributes directly to peptidyl transferase center function and the translation elongation cycle [PMID:19647033, PMID:30550856]. Its conserved GGQ-containing motif (47GFGGQTK53) contacts the CCA end of P-site tRNA [PMID:19647033], and mutations in or adjacent to this minidomain impair poly(Phe) synthesis and peptidyl transferase activity toward puromycin, with a shifted side-chain pKa indicating a catalytic contribution [PMID:30550856]. Within the human ribosome, cooperative interactions among residues Q45, Q51, and K53 of the conserved QSGYGGQTK motif are specifically required for dissociation of deacylated tRNA from the E site during elongation, independent of subunit assembly [PMID:37716853], and the protein binds tRNA and 28S rRNA with high affinity while engaging the methylated GGQ motif of the termination factor eRF1 through methyl-group contacts [PMID:28567122]. RPL36A is post-translationally modified: its N-terminal methionine is removed [PMID:3396452], and the lysine methyltransferase SET7/9 binds eL42 via its MORN domain and methylates it at Lys53, Lys80, and Lys100, thereby modulating global translation [PMID:31751593]. Beyond its core ribosomal role, RPL36A can selectively influence translation of specific mRNAs such as Hsp90α under stress [PMID:28090422] and acts as a proliferative factor in cancer, promoting cell cycling and colony formation [PMID:14752831] and operating upstream of the MAPK/ERK pathway to sustain pERK, c-Myc, and ELK1 expression in colorectal cancer cells [PMID:39489085]. The protein localizes to nucleoli through determinants in its N-terminal domain [PMID:14752831].","teleology":[{"year":1988,"claim":"Established the primary structure and conservation of the protein, defining it as a member of the L44e family near the peptidyl transferase center and documenting N-terminal methionine removal as a constitutive modification.","evidence":"cDNA and N-terminal protein sequencing of rat L36a with Southern hybridization for gene copy number","pmids":["3396452"],"confidence":"Medium","gaps":["No direct demonstration of catalytic role in rat protein","Functional consequence of Met removal not tested"]},{"year":2004,"claim":"Connected the protein to subcellular targeting and to a cellular phenotype, showing nucleolar localization driven by N-terminal determinants and a proliferative role in cancer cells.","evidence":"Ectopic over-expression with localization imaging and domain mapping, plus antisense knockdown with colony formation, proliferation, and cell cycle assays in liver cells","pmids":["14752831"],"confidence":"Medium","gaps":["Does not distinguish ribosomal from extra-ribosomal mechanism of proliferation","No molecular pathway identified at this stage"]},{"year":2009,"claim":"Placed the protein physically at the tRNA substrate site, showing it contacts the CCA end of P-site tRNA in human 80S ribosomes.","evidence":"Zero-length periodate-oxidized tRNA cross-linking to human 80S ribosomes with competition and MS identification","pmids":["19647033"],"confidence":"Medium","gaps":["Cross-link reported for the RPL36AL paralog","Does not establish catalytic contribution, only proximity"]},{"year":2016,"claim":"Linked eL42 levels to selective translation, showing it can favor Hsp90α mRNA translation while global translation is suppressed during heat stress.","evidence":"Polysome profiling and eL42 level manipulation in rhabdomyosarcoma cells with 17-AAG sensitivity readout","pmids":["28090422"],"confidence":"Medium","gaps":["Mechanism of mRNA selectivity unknown","Direct binding of eL42 to Hsp90α mRNA not shown"]},{"year":2017,"claim":"Resolved the molecular basis of eL42 participation in translation termination, mapping methylated Gln-51/Lys-53 contacts to the methylated GGQ of eRF1 and quantifying tRNA and rRNA binding.","evidence":"Surface plasmon resonance binding of recombinant wild-type and mutant eL42 against eRF1, tRNA, and 28S rRNA","pmids":["28567122"],"confidence":"Medium","gaps":["In vitro binding only; functional termination consequence not measured","Methylation state of recombinant protein in assay not fully defined"]},{"year":2017,"claim":"Implicated the protein in CAP-independent translation and stress-induced HSP expression in yeast.","evidence":"Gene deletion and genetic analysis in S. cerevisiae monitoring HSP82/HSC82 and CAP-independent translation under acetic acid and heat stress","pmids":["29158977"],"confidence":"Low","gaps":["Genetic observation with limited mechanistic resolution on pathway position","Direct role of eL42 versus DOM34 not separated"]},{"year":2018,"claim":"Demonstrated that the GGQ minidomain is catalytically required, with genetic complementation failure and a depressed side-chain pKa pointing to direct involvement in peptidyl transfer.","evidence":"Site-directed mutagenesis, genetic complementation, poly(Phe) and puromycin assays, and pKa measurement in S. pombe","pmids":["30550856"],"confidence":"High","gaps":["Catalytic mechanism in human ribosome inferred from yeast ortholog","No high-resolution structure of the catalytic geometry"]},{"year":2019,"claim":"Identified SET7/9 as a methyltransferase for eL42 and tied this modification to control of global translation.","evidence":"Yeast two-hybrid, co-IP, GST pulldown, methylation-site mutagenesis, and puromycin incorporation assay","pmids":["31751593"],"confidence":"High","gaps":["Quantitative stoichiometry of methylation in vivo unknown","How methylation alters ribosome behavior mechanistically not resolved"]},{"year":2021,"claim":"Connected RPL36A to the DNA damage and apoptotic response, showing knockdown increases radiosensitivity in oral squamous cell carcinoma.","evidence":"siRNA knockdown with irradiation, DNA damage markers, cell cycle flow cytometry, and apoptosis assays in OSCC lines","pmids":["34830778"],"confidence":"Medium","gaps":["Whether the effect is via translation or an extra-ribosomal route is unresolved","No direct molecular target of the radiosensitization identified"]},{"year":2022,"claim":"Proposed eL42 incorporation level as a source of ribosome heterogeneity, with paralog deletion phenotypes tracing to Rpl42p abundance rather than sequence.","evidence":"Paralog gene deletion, polysome profiling, Western blotting of translating fractions, and epistasis in S. pombe","pmids":["35954225"],"confidence":"Medium","gaps":["Functional consequence of heterogeneous eL42 content on specific mRNAs unknown","Relevance to human ribosomes untested"]},{"year":2023,"claim":"Defined a specific elongation step for eL42 in human ribosomes, showing cooperative Q45/Q51/K53 interactions are required for deacylated tRNA release from the E site without affecting subunit assembly.","evidence":"Conserved-motif mutagenesis in FLAG-tagged eL42 in HEK293T cells with polysome profiling, tRNA binding, and peptidyl transferase assays","pmids":["37716853"],"confidence":"High","gaps":["Structural basis of the E-site coupling not visualized","Interplay with methylation state of these residues not tested"]},{"year":2024,"claim":"Placed RPL36A upstream of MAPK/ERK signaling in cancer, with knockdown reducing pERK/c-Myc/ELK1 and ERK activation rescuing the phenotype.","evidence":"siRNA knockdown in CRC lines with Western blotting, ERK-activator rescue, and xenograft growth assay","pmids":["39489085"],"confidence":"Medium","gaps":["Direct molecular link between RPL36A and ERK activation not defined","Whether the effect requires ribosomal function is unclear"]},{"year":null,"claim":"How the catalytic, termination, methylation, mRNA-selectivity, and extra-ribosomal signaling functions are integrated, and the structural basis of eL42's E-site role, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of eL42 in the catalytically engaged ribosome","Mechanism coupling ribosomal residence to MAPK/ERK activation undefined","Physiological role of methylation-dependent eRF1 interaction in cells not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2,4]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[5,10]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[5,10]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[0]},{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[2,10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[11]}],"complexes":["60S ribosomal subunit"],"partners":["ERF1","SETD7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P83881","full_name":"Large ribosomal subunit protein eL42","aliases":["60S ribosomal protein L36a","60S ribosomal protein L44","Cell growth-inhibiting gene 15 protein","Cell migration-inducing gene 6 protein"],"length_aa":106,"mass_kda":12.4,"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/P83881/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RPL36A","classification":"Common Essential","n_dependent_lines":357,"n_total_lines":380,"dependency_fraction":0.9394736842105263},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RPL36A","total_profiled":1310},"omim":[{"mim_id":"617649","title":"UBIQUITIN-CONJUGATING ENZYME E2 O; UBE2O","url":"https://www.omim.org/entry/617649"},{"mim_id":"300902","title":"RIBOSOMAL PROTEIN L36A; RPL36A","url":"https://www.omim.org/entry/300902"},{"mim_id":"180469","title":"RIBOSOMAL PROTEIN L36A-LIKE; RPL36AL","url":"https://www.omim.org/entry/180469"},{"mim_id":"180466","title":"RIBOSOMAL PROTEIN L19; RPL19","url":"https://www.omim.org/entry/180466"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RPL36A"},"hgnc":{"alias_symbol":["L36A","eL42"],"prev_symbol":["RPL44"]},"alphafold":{"accession":"P83881","domains":[{"cath_id":"3.10.450.80","chopping":"1-28_62-98","consensus_level":"medium","plddt":94.4311,"start":1,"end":98}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P83881","model_url":"https://alphafold.ebi.ac.uk/files/AF-P83881-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P83881-F1-predicted_aligned_error_v6.png","plddt_mean":94.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RPL36A","jax_strain_url":"https://www.jax.org/strain/search?query=RPL36A"},"sequence":{"accession":"P83881","fasta_url":"https://rest.uniprot.org/uniprotkb/P83881.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P83881/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P83881"}},"corpus_meta":[{"pmid":"14752831","id":"PMC_14752831","title":"Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma.","date":"2004","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/14752831","citation_count":88,"is_preprint":false},{"pmid":"24814782","id":"PMC_24814782","title":"Abiotic stress resistance, a novel moonlighting function of ribosomal protein RPL44 in the halophilic fungus Aspergillus glaucus.","date":"2014","source":"Applied and environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/24814782","citation_count":41,"is_preprint":false},{"pmid":"19647033","id":"PMC_19647033","title":"The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA.","date":"2009","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/19647033","citation_count":25,"is_preprint":false},{"pmid":"31751593","id":"PMC_31751593","title":"SET7/9 interacts and methylates the ribosomal protein, eL42 and regulates protein synthesis.","date":"2019","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/31751593","citation_count":14,"is_preprint":false},{"pmid":"29158977","id":"PMC_29158977","title":"The sensitivity of the yeast, Saccharomyces cerevisiae, to acetic acid is influenced by DOM34 and RPL36A.","date":"2017","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/29158977","citation_count":14,"is_preprint":false},{"pmid":"34830778","id":"PMC_34830778","title":"Characterization of Recurrent Relevant Genes Reveals a Novel Role of RPL36A in Radioresistant Oral Squamous Cell Carcinoma.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34830778","citation_count":12,"is_preprint":false},{"pmid":"3396452","id":"PMC_3396452","title":"Primary structure of rat ribosomal protein L36a.","date":"1988","source":"DNA (Mary Ann Liebert, Inc.)","url":"https://pubmed.ncbi.nlm.nih.gov/3396452","citation_count":11,"is_preprint":false},{"pmid":"28090422","id":"PMC_28090422","title":"Elevated levels of ribosomal proteins eL36 and eL42 control expression of Hsp90 in rhabdomyosarcoma.","date":"2016","source":"Translation (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/28090422","citation_count":6,"is_preprint":false},{"pmid":"30550856","id":"PMC_30550856","title":"Ribosomal protein eL42 contributes to the catalytic activity of the yeast ribosome at the elongation step of translation.","date":"2018","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/30550856","citation_count":5,"is_preprint":false},{"pmid":"35954225","id":"PMC_35954225","title":"Differential Paralog-Specific Expression of Multiple Small Subunit Proteins Cause Variations in Rpl42/eL42 Incorporation in Ribosome in Fission Yeast.","date":"2022","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/35954225","citation_count":5,"is_preprint":false},{"pmid":"39489085","id":"PMC_39489085","title":"RPL36A activates ERK pathway and promotes colorectal cancer growth.","date":"2024","source":"Translational oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39489085","citation_count":3,"is_preprint":false},{"pmid":"20416165","id":"PMC_20416165","title":"[Proliferation promotion and apoptotic inhibition effects of ribosomal protein RPL36A small interference RNA on U937 cells].","date":"2010","source":"Zhongguo shi yan xue ye xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/20416165","citation_count":3,"is_preprint":false},{"pmid":"28567122","id":"PMC_28567122","title":"A Functional Role for the Monomethylated Gln-51 and Lys-53 Residues of the 49GGQTK53 Motif of eL42 from Human 80S Ribosomes.","date":"2017","source":"The open biochemistry journal","url":"https://pubmed.ncbi.nlm.nih.gov/28567122","citation_count":2,"is_preprint":false},{"pmid":"11328654","id":"PMC_11328654","title":"Molecular cloning and complete cDNA sequences of the ribosomal proteins rpl34 and rpl44 from Aedes triseriatus mosquitoes.","date":"2000","source":"DNA sequence : the journal of DNA sequencing and mapping","url":"https://pubmed.ncbi.nlm.nih.gov/11328654","citation_count":1,"is_preprint":false},{"pmid":"37716853","id":"PMC_37716853","title":"Functional involvement of a conserved motif in the middle region of the human ribosomal protein eL42 in translation.","date":"2023","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/37716853","citation_count":1,"is_preprint":false},{"pmid":"7941754","id":"PMC_7941754","title":"RPL44 and RPL44', encoding acidic ribosomal phosphoproteins YP2 alpha(L44) and YP1 beta(L44'), are adjacent to rig and STF1 on Saccharomyces cerevisiae chromosomes XV and IV respectively.","date":"1994","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/7941754","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9229,"output_tokens":3225,"usd":0.038031,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10675,"output_tokens":3758,"usd":0.073663,"stage2_stop_reason":"end_turn"},"total_usd":0.111694,"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\": 2004,\n      \"finding\": \"Over-expressed L36a/RPL36A ribosomal protein localizes to the nucleoli of cells, and this localization is controlled by the N-terminal or the internal tetrapeptide consensus sequence with its adjacent N-terminal domain.\",\n      \"method\": \"Ectopic over-expression with subcellular localization imaging (nucleolar localization assay); N-terminal domain mapping\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment with domain mapping in single lab, but no functional reconstitution or structural validation\",\n      \"pmids\": [\"14752831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Over-expression of RPL36A leads to enhanced colony formation and cell proliferation via accelerated cell cycling; antisense cDNA effectively reversed these effects, establishing a direct role for RPL36A in promoting cellular proliferation.\",\n      \"method\": \"Ectopic over-expression and antisense knockdown with colony formation assay, proliferation assay, and cell cycle analysis\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function with defined cellular phenotypes, single lab\",\n      \"pmids\": [\"14752831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The large subunit ribosomal protein RPL36A-like (RPL36AL/eL42) contacts the CCA end of P-site bound tRNA in human 80S ribosomes, placing it at or near the binding site for the tRNA substrate of the peptidyl transferase reaction.\",\n      \"method\": \"Periodate-oxidized tRNA (zero-length affinity labeling reagent) cross-linking to human 80S ribosomes, with competition by intact tRNA and protein identification by mass spectrometry\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro affinity labeling with direct chemical cross-linking and MS identification, competition assay, single lab\",\n      \"pmids\": [\"19647033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"Rat ribosomal protein L36a (homolog of human RPL36A) contains 105 amino acids (N-terminal Met removed post-translationally) and is homologous to human HL44 and yeast protein 44, suggesting conservation of the L44e family at the peptidyl transferase center.\",\n      \"method\": \"cDNA sequencing of recombinant clone, N-terminal amino acid sequencing, Southern hybridization for gene copy number\",\n      \"journal\": \"DNA (Mary Ann Liebert, Inc.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein sequencing confirmed by cDNA sequence, single lab; N-terminal methionine removal established as PTM\",\n      \"pmids\": [\"3396452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The monomethylated Gln-51 and Lys-53 residues within the 47GFGGQTK53 motif of human eL42 (RPL36A) mediate interaction with the methylated GGQ motif of eRF1 via hydrophobic contacts between methyl groups; eL42 also interacts with tRNA (nanomolar KD) and 28S rRNA (picomolar KD) through strong binding affinities.\",\n      \"method\": \"Biacore surface plasmon resonance binding assay using recombinant wild-type and mutant eL42 proteins (GGQTK deletion, Q51E, K53Q, Q51A/K53A double mutant) against eRF1, tRNA, and 28S rRNA\",\n      \"journal\": \"The open biochemistry journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assay with site-directed mutagenesis, single lab, multiple mutants tested\",\n      \"pmids\": [\"28567122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The GGQ minidomain and neighboring region of eL42 (RPL36A ortholog) is critical for ribosomal function in S. pombe; mutations within or adjacent to the GGQ minidomain fail to complement wild-type eL42 and cause growth defects, reduced poly(Phe) synthesis, and reduced peptidyl transferase activity toward puromycin. A pKa of 6.95 was measured for the side chain of Lys-55/Arg-55, substantially less than a free Lys or Arg, indicating involvement in catalysis.\",\n      \"method\": \"Site-directed mutagenesis, genetic complementation in S. pombe, in vitro poly(Phe) synthesis assay, peptidyl transferase (puromycin) assay, pKa measurement\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal in vitro and in vivo methods (mutagenesis, genetic complementation, biochemical assays), rigorous mechanistic dissection of catalytic contribution\",\n      \"pmids\": [\"30550856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The lysine methyltransferase SET7/9 interacts with eL42 (RPL36A) via its N-terminal MORN domain, and methylates eL42 at three lysines (Lys53, Lys80, Lys100); SET7/9-mediated methylation of eL42 affects global protein translation.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, GST pulldown, site-directed mutagenesis of methylation sites, puromycin incorporation assay for translation\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation with mutagenesis, reciprocal co-IP and GST pulldown, functional translation assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31751593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Elevated levels of eL42 (RPL36A) modulate Hsp90α expression under both normal and heat shock conditions in rhabdomyosarcoma cells; polysome profiling showed selective translation of Hsp90α mRNA while global translation is inhibited during heat stress.\",\n      \"method\": \"Polysome profiling, manipulation of eL42 levels in rhabdomyosarcoma cells with measurement of Hsp90α expression and sensitivity to Hsp90 inhibitor 17-AAG\",\n      \"journal\": \"Translation (Austin, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — polysome profiling with functional drug sensitivity assay, single lab\",\n      \"pmids\": [\"28090422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RPL36A and DOM34 influence CAP-independent translation in yeast and affect expression of HSP82 and HSC82 heat shock proteins in response to acetic acid stress.\",\n      \"method\": \"Gene deletion/genetic analysis in S. cerevisiae, monitoring of HSP protein expression and CAP-independent translation under acetic acid and heat stress\",\n      \"journal\": \"PeerJ\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic observations, single lab, limited mechanistic resolution on pathway position\",\n      \"pmids\": [\"29158977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Knockdown of RPL36A in oral squamous cell carcinoma cells increased radiosensitivity by sensitizing cells to DNA damage, promoting G2/M cell cycle arrest, and augmenting irradiation-induced apoptosis.\",\n      \"method\": \"siRNA knockdown of RPL36A in OSCC cell lines, irradiation, measurement of DNA damage markers, cell cycle analysis by flow cytometry, apoptosis assay\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotypes (DNA damage, cell cycle, apoptosis), single lab\",\n      \"pmids\": [\"34830778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cooperative interactions involving eL42 (RPL36A) residues Q45, Q51, and K53 within the conserved QSGYGGQTK motif are critical for the human ribosome elongation cycle specifically at the step of deacylated tRNA dissociation from the E site; double substitutions at positions 45+51 or 45+53 decrease polysomes without affecting 60S or 80S assembly.\",\n      \"method\": \"Site-directed mutagenesis of conserved motif residues in FLAG-tagged eL42, expression in HEK293T cells, polysome profiling with Western blotting, tRNA binding assay, peptidyl transferase activity assay\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with multiple orthogonal assays (polysome profiling, tRNA binding, enzymatic activity) in human cells, single lab\",\n      \"pmids\": [\"37716853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RPL36A depletion in colorectal cancer cells diminishes phosphorylated ERK levels and subsequently reduces expression of c-Myc and ELK1; the tumor-suppressive effects of RPL36A knockdown are negated by an ERK activator, placing RPL36A upstream of the MAPK/ERK pathway.\",\n      \"method\": \"siRNA knockdown of RPL36A in CRC cell lines, Western blotting for pERK/c-Myc/ELK1, rescue with ERK activator, tumor xenograft growth assay\",\n      \"journal\": \"Translational oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via rescue experiment with ERK activator, loss-of-function with defined signaling phenotype, single lab\",\n      \"pmids\": [\"39489085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In S. pombe, the level of Rpl42p (eL42/RPL36A ortholog) in actively translating ribosomes varies depending on which 40S ribosomal protein paralog is present, identifying variation in eL42 incorporation as a potential form of ribosome heterogeneity; phenotypic differences of rps8 paralog deletions reside in Rpl42p level variation rather than in protein sequence differences between the Rps8 paralogs.\",\n      \"method\": \"Genetic deletion of paralog genes in fission yeast, polysome profiling, Western blotting to quantify Rpl42p in translating fractions, epistasis analysis\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus polysome fractionation and Western blotting, single lab\",\n      \"pmids\": [\"35954225\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RPL36A (eL42) is a component of the large (60S) ribosomal subunit whose conserved GGQ-containing motif (47GFGGQTK53) directly contacts the CCA end of P-site tRNA, contributes to peptidyl transferase activity and the elongation cycle (specifically deacylated tRNA release from the E site), is post-translationally methylated by SET7/9 at Lys53/Lys80/Lys100 (which affects global translation), interacts with eRF1 via methylated GGQ contacts during termination, is subject to N-terminal methionine removal, localizes to nucleoli via its N-terminal domain, and extra-ribosomally activates the MAPK/ERK signaling pathway to promote proliferation in cancer contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RPL36A (eL42) is a component of the large (60S) ribosomal subunit that contributes directly to peptidyl transferase center function and the translation elongation cycle [#2, #5]. Its conserved GGQ-containing motif (47GFGGQTK53) contacts the CCA end of P-site tRNA [#2], and mutations in or adjacent to this minidomain impair poly(Phe) synthesis and peptidyl transferase activity toward puromycin, with a shifted side-chain pKa indicating a catalytic contribution [#5]. Within the human ribosome, cooperative interactions among residues Q45, Q51, and K53 of the conserved QSGYGGQTK motif are specifically required for dissociation of deacylated tRNA from the E site during elongation, independent of subunit assembly [#10], and the protein binds tRNA and 28S rRNA with high affinity while engaging the methylated GGQ motif of the termination factor eRF1 through methyl-group contacts [#4]. RPL36A is post-translationally modified: its N-terminal methionine is removed [#3], and the lysine methyltransferase SET7/9 binds eL42 via its MORN domain and methylates it at Lys53, Lys80, and Lys100, thereby modulating global translation [#6]. Beyond its core ribosomal role, RPL36A can selectively influence translation of specific mRNAs such as Hsp90\\u03b1 under stress [#7] and acts as a proliferative factor in cancer, promoting cell cycling and colony formation [#1] and operating upstream of the MAPK/ERK pathway to sustain pERK, c-Myc, and ELK1 expression in colorectal cancer cells [#11]. The protein localizes to nucleoli through determinants in its N-terminal domain [#0].\",\n  \"teleology\": [\n    {\n      \"year\": 1988,\n      \"claim\": \"Established the primary structure and conservation of the protein, defining it as a member of the L44e family near the peptidyl transferase center and documenting N-terminal methionine removal as a constitutive modification.\",\n      \"evidence\": \"cDNA and N-terminal protein sequencing of rat L36a with Southern hybridization for gene copy number\",\n      \"pmids\": [\"3396452\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct demonstration of catalytic role in rat protein\", \"Functional consequence of Met removal not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Connected the protein to subcellular targeting and to a cellular phenotype, showing nucleolar localization driven by N-terminal determinants and a proliferative role in cancer cells.\",\n      \"evidence\": \"Ectopic over-expression with localization imaging and domain mapping, plus antisense knockdown with colony formation, proliferation, and cell cycle assays in liver cells\",\n      \"pmids\": [\"14752831\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not distinguish ribosomal from extra-ribosomal mechanism of proliferation\", \"No molecular pathway identified at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Placed the protein physically at the tRNA substrate site, showing it contacts the CCA end of P-site tRNA in human 80S ribosomes.\",\n      \"evidence\": \"Zero-length periodate-oxidized tRNA cross-linking to human 80S ribosomes with competition and MS identification\",\n      \"pmids\": [\"19647033\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cross-link reported for the RPL36AL paralog\", \"Does not establish catalytic contribution, only proximity\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked eL42 levels to selective translation, showing it can favor Hsp90\\u03b1 mRNA translation while global translation is suppressed during heat stress.\",\n      \"evidence\": \"Polysome profiling and eL42 level manipulation in rhabdomyosarcoma cells with 17-AAG sensitivity readout\",\n      \"pmids\": [\"28090422\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of mRNA selectivity unknown\", \"Direct binding of eL42 to Hsp90\\u03b1 mRNA not shown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the molecular basis of eL42 participation in translation termination, mapping methylated Gln-51/Lys-53 contacts to the methylated GGQ of eRF1 and quantifying tRNA and rRNA binding.\",\n      \"evidence\": \"Surface plasmon resonance binding of recombinant wild-type and mutant eL42 against eRF1, tRNA, and 28S rRNA\",\n      \"pmids\": [\"28567122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro binding only; functional termination consequence not measured\", \"Methylation state of recombinant protein in assay not fully defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Implicated the protein in CAP-independent translation and stress-induced HSP expression in yeast.\",\n      \"evidence\": \"Gene deletion and genetic analysis in S. cerevisiae monitoring HSP82/HSC82 and CAP-independent translation under acetic acid and heat stress\",\n      \"pmids\": [\"29158977\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Genetic observation with limited mechanistic resolution on pathway position\", \"Direct role of eL42 versus DOM34 not separated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated that the GGQ minidomain is catalytically required, with genetic complementation failure and a depressed side-chain pKa pointing to direct involvement in peptidyl transfer.\",\n      \"evidence\": \"Site-directed mutagenesis, genetic complementation, poly(Phe) and puromycin assays, and pKa measurement in S. pombe\",\n      \"pmids\": [\"30550856\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism in human ribosome inferred from yeast ortholog\", \"No high-resolution structure of the catalytic geometry\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified SET7/9 as a methyltransferase for eL42 and tied this modification to control of global translation.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, GST pulldown, methylation-site mutagenesis, and puromycin incorporation assay\",\n      \"pmids\": [\"31751593\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative stoichiometry of methylation in vivo unknown\", \"How methylation alters ribosome behavior mechanistically not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected RPL36A to the DNA damage and apoptotic response, showing knockdown increases radiosensitivity in oral squamous cell carcinoma.\",\n      \"evidence\": \"siRNA knockdown with irradiation, DNA damage markers, cell cycle flow cytometry, and apoptosis assays in OSCC lines\",\n      \"pmids\": [\"34830778\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the effect is via translation or an extra-ribosomal route is unresolved\", \"No direct molecular target of the radiosensitization identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Proposed eL42 incorporation level as a source of ribosome heterogeneity, with paralog deletion phenotypes tracing to Rpl42p abundance rather than sequence.\",\n      \"evidence\": \"Paralog gene deletion, polysome profiling, Western blotting of translating fractions, and epistasis in S. pombe\",\n      \"pmids\": [\"35954225\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of heterogeneous eL42 content on specific mRNAs unknown\", \"Relevance to human ribosomes untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined a specific elongation step for eL42 in human ribosomes, showing cooperative Q45/Q51/K53 interactions are required for deacylated tRNA release from the E site without affecting subunit assembly.\",\n      \"evidence\": \"Conserved-motif mutagenesis in FLAG-tagged eL42 in HEK293T cells with polysome profiling, tRNA binding, and peptidyl transferase assays\",\n      \"pmids\": [\"37716853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the E-site coupling not visualized\", \"Interplay with methylation state of these residues not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed RPL36A upstream of MAPK/ERK signaling in cancer, with knockdown reducing pERK/c-Myc/ELK1 and ERK activation rescuing the phenotype.\",\n      \"evidence\": \"siRNA knockdown in CRC lines with Western blotting, ERK-activator rescue, and xenograft growth assay\",\n      \"pmids\": [\"39489085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between RPL36A and ERK activation not defined\", \"Whether the effect requires ribosomal function is unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the catalytic, termination, methylation, mRNA-selectivity, and extra-ribosomal signaling functions are integrated, and the structural basis of eL42's E-site role, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of eL42 in the catalytically engaged ribosome\", \"Mechanism coupling ribosomal residence to MAPK/ERK activation undefined\", \"Physiological role of methylation-dependent eRF1 interaction in cells not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [5, 10]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [5, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [2, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-72766\", \"supporting_discovery_ids\": [5, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"complexes\": [\"60S ribosomal subunit\"],\n    \"partners\": [\"ERF1\", \"SETD7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}