{"gene":"RPL4","run_date":"2026-06-10T07:46:26","timeline":{"discoveries":[{"year":1980,"finding":"E. coli ribosomal protein L4 autogenously regulates the S10 operon by inhibiting translation of the promoter-proximal proteins (S10, L3, L4, L23, L2) in vitro; no other S10 operon protein caused selective inhibition, and L4 did not inhibit other operons.","method":"Lambda fus3 DNA-directed in vitro protein synthesis system with purified L4 protein addition","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified protein, replicated in companion paper (PMID:6157482)","pmids":["6996835"],"is_preprint":false},{"year":1980,"finding":"E. coli ribosomal protein L4, when overproduced in vivo, reduces mRNA synthesis from at least four genes of the S10 operon, identifying L4 as the sole autogenous regulator among eleven S10 operon products.","method":"In vivo oversynthesis of individual S10 operon ribosomal proteins with measurement of individual protein synthesis rates","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic overexpression, replicated alongside in vitro reconstitution (PMID:6996835)","pmids":["6157482"],"is_preprint":false},{"year":1975,"finding":"Ribosomal protein L4 is the primary RNA-crosslinked protein in the E. coli 50S subunit upon UV irradiation, demonstrating direct RNA-protein contact in the ribosome.","method":"UV cross-linking of 50S subunits followed by Sarkosyl gel analysis, 2D electrophoresis, and immunological identification with protein-specific antisera","journal":"Molecular & general genetics : MGG","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical cross-linking with multiple orthogonal verification methods","pmids":["814400"],"is_preprint":false},{"year":1980,"finding":"The UV cross-link between L4 and 23S rRNA is located at tyrosine-35 of L4 and uridine-615 of 23S rRNA, establishing the precise RNA-protein contact site.","method":"Isolation of cross-linked L4-oligonucleotide complexes, successive protease and nuclease digestions, 2D gel electrophoresis, oligonucleotide analysis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — precise biochemical mapping with multiple orthogonal digestion/analysis methods","pmids":["6998491"],"is_preprint":false},{"year":1988,"finding":"L4 is cross-linked to nucleotides 320–325 of E. coli 23S rRNA when 50S subunits are treated with 2-iminothiolane followed by UV irradiation, identifying an additional RNA contact site.","method":"Chemical cross-linking (2-iminothiolane + UV), affinity chromatography with antibodies, partial RNA digestion, complex isolation","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous biochemical method but single lab, single study","pmids":["3278299"],"is_preprint":false},{"year":1990,"finding":"L4 stimulates premature transcription termination approximately 140 bases from the transcription start site within the S10 operon leader, upstream of the first structural gene, by a mechanism independent of its inhibition of translation.","method":"In vivo mapping of 5′ and 3′ ends of newly synthesized RNA; deletion of sequences downstream of termination site","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo mapping plus genetic deletion to dissociate transcriptional from translational control, replicated in vitro (PMID:2157208)","pmids":["1692593"],"is_preprint":false},{"year":1990,"finding":"L4 stimulates termination of transcription at the S10 attenuator in vitro, but RNA polymerase requires NusA to terminate at this site; L4 increases termination efficiency at the NusA-dependent pause site.","method":"Purified in vitro transcription system with purified L4 and NusA proteins","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro transcription with purified components, replicated across multiple papers","pmids":["2157208"],"is_preprint":false},{"year":1992,"finding":"NusA is required for RNA polymerase pausing at the S10 attenuation site; L4 further stabilizes the NusA-modified paused complex. These two activities require genetically separable regions of the S10 leader RNA.","method":"In vitro transcription with purified components; isolation of paused complexes; genetic deletion analysis of leader RNA","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro with purified proteins plus genetic dissection","pmids":["1285127"],"is_preprint":false},{"year":1995,"finding":"The upper stem-loop of the attenuator hairpin is the major determinant for NusA-dependent pausing; upstream hairpin and the ascending side of the attenuator are required for L4 stabilization of the paused complex; NusA is not absolutely required for initial RNA polymerase pausing at low UTP concentration, but is required for L4 to stabilize the pause.","method":"Genetic and antisense oligonucleotide competition with purified in vitro transcription system; isolation of paused complexes","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution combined with genetic and oligonucleotide-based dissection","pmids":["7844821","7531246"],"is_preprint":false},{"year":1999,"finding":"E. coli L4 binds directly and specifically to the S10 mRNA leader in vitro; the binding site is a small hairpin structure within the leader but a 64-nucleotide sequence is required for full interaction. Phosphorothioate footprinting shows structural mimicry between the mRNA and rRNA binding sites in three dimensions.","method":"In vitro binding assays, competition with 23S rRNA L4-binding site, phosphorothioate footprinting, mutational analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro binding reconstitution with footprinting and mutagenesis in one study","pmids":["12738792"],"is_preprint":false},{"year":1996,"finding":"L4 uses non-identical determinants for ribosome assembly and autogenous S10 operon regulation: mutations were isolated that eliminate autogenous control but allow ribosome assembly, and vice versa, showing the two RNA-binding interfaces are distinct.","method":"Genetic isolation of L4 mutants; functional assays for ribosome assembly and autogenous control","journal":"RNA","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic dissection with multiple separable mutants, two orthogonal functional readouts","pmids":["8846294"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of Thermotoga maritima L4 at 1.7 Å resolution reveals an alternating α/β fold with a large disordered loop; two separate RNA-binding sites are identified: an N-terminal site (disordered loop with flanking helices) for rRNA binding and a C-terminal site (two non-consecutive helices) candidate for S10 mRNA leader interaction; a C-terminal protein-binding interface is also identified.","method":"X-ray crystallography at 1.7 Å resolution","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mapping of binding sites","pmids":["10698923"],"is_preprint":false},{"year":1999,"finding":"Erythromycin resistance mutations in ribosomal protein L4 (but not L22) of E. coli result in decreased peptide-bond-forming activity and perturb the conformation of 23S rRNA nucleotides in domains II, III, and V, without affecting the A2058 region—indicating L4 mutations act by altering rRNA higher-order structure.","method":"Chemical modification/probing of 23S rRNA structure in ribosomes from resistance mutants; multiple chemical probes at multiple nucleotide positions","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct structural probing with multiple reagents on defined mutant ribosomes","pmids":["10369764"],"is_preprint":false},{"year":2004,"finding":"L4 mutant ribosomes with altered L4 protein show increased frameshifting, missense decoding, stop codon readthrough, non-AUG initiation, and mutant initiator tRNA utilization, demonstrating that L4 mutations in the 50S subunit also alter 30S subunit structure and function upon association.","method":"In vivo translational fidelity assays (frameshifting, readthrough, missense) in E. coli L4 and L22 mutant strains; antibiotic interaction assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vivo functional assays in defined mutant strains","pmids":["15509870"],"is_preprint":false},{"year":2007,"finding":"Eight new L4 mutations in the tentacle region of E. coli L4 confer erythromycin resistance; all reduce in vivo peptide-chain elongation rates and increase precursor 23S rRNA levels; large insertions in L4 cause accumulation of abnormal ribosomal subunits, highlighting L4's role in ribosome assembly and function.","method":"In vivo isolation of erythromycin-resistant mutants; sequencing; growth rate measurements; ribosome profiling; rRNA precursor analysis","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple mutants characterized with multiple orthogonal functional readouts","pmids":["17956547"],"is_preprint":false},{"year":2009,"finding":"E. coli ribosomal protein L4 interacts with RNase E outside its catalytic domain and inhibits RNase E endoribonucleolytic activity in vitro; in vivo, ectopic L4 stabilizes RNase E-targeted mRNAs and stabilizes an antisense regulatory RNA controlling plasmid replication; L4 overexpression broadens mRNA stability for stress-response transcripts.","method":"Affinity purification, immunoprecipitation, E. coli two-hybrid screening, in vitro cleavage assays, mRNA half-life measurements, DNA microarray","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including in vitro reconstitution and in vivo mRNA stability assays","pmids":["19144914"],"is_preprint":false},{"year":2015,"finding":"Eukaryotic Rpl4 (yeast) requires a dedicated assembly chaperone Acl4, which binds the universally conserved internal loop of newly synthesized Rpl4 via a superhelical TPR domain, preventing premature rRNA insertion. Rpl4's eukaryote-specific extension makes distinct interactions with the 60S surface (including a co-evolved site on Rpl18) that orchestrate Acl4 release and Rpl4 pre-ribosome incorporation.","method":"Yeast genetics, biochemical pulldowns, co-IP, mutational analysis of RpL4 and RpL18 contact sites, ribosome assembly assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetics, biochemistry, mutagenesis of interacting surfaces) in a single rigorous study","pmids":["25936803"],"is_preprint":false},{"year":2015,"finding":"Acl4 acts as dedicated chaperone that binds the long internal loop of Rpl4 (yeast), accompanies it from the cytoplasm to the nucleus, and is required for 60S subunit production; both the internal loop and C-terminal eukaryote-specific extension are essential for Rpl4 function; Rpl4 contains at least five nuclear localization signals.","method":"Yeast genetics (slow-growth and 60S deficiency phenotypes), biochemical co-fractionation, co-translational capture assays, localization studies","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic and biochemical methods replicating findings of PMID:25936803","pmids":["26447800"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of yeast Rpl4 bound to its chaperone Acl4 reveals extensive interactions sequestering 70 exposed residues of the extended Rpl4 loop; the eukaryote-specific Rpl4 extension harbors overlapping binding sites for Acl4 and nuclear transport factor Kap104, enabling continuous protection from cellular degradation machinery.","method":"X-ray crystallography of Rpl4–Acl4 complex; biochemical binding assays for Kap104 competition; degradation assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus biochemical validation of overlapping binding sites","pmids":["28148929"],"is_preprint":false},{"year":2014,"finding":"In yeast, deletion of Rpl4's conserved internal loop impairs growth and reduces large ribosomal subunit levels, affecting later steps in assembly; depletion of full Rpl4 blocks early assembly steps, revealing two distinct roles for L4 in ribosome biogenesis. Deletion of the entire eukaryote-specific C-terminal extension has no effect on viability or 60S production.","method":"Yeast genetics (conditional depletion, deletion mutants), ribosome profiling, polysome analysis, pulse-chase labeling","journal":"RNA","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple deletion mutants with defined assembly phenotypes and orthogonal ribosome analyses","pmids":["25246649"],"is_preprint":false},{"year":2016,"finding":"In E. coli, deletion of the uL4 loop causes cold-sensitive ribosome assembly defects and accumulation of immature particles; the uL4 loop deletion also impairs response to the cmlA(crb) translation pause peptide but not to the secM pause peptide, while uL22 loop deletion affects different assembly intermediates.","method":"Genetic analysis using strains expressing only loop-deleted uL4; ribosome sedimentation, rRNA processing analysis, in vivo pausing assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — defined genetic strains with multiple orthogonal assays for assembly and translation function","pmids":["27257065"],"is_preprint":false},{"year":2013,"finding":"In yeast, ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 all contact the signal anchor of nascent chains within the exit tunnel; Rpl4 uniquely contacts nascent chain residues throughout the entire ribosomal tunnel length, whereas Rpl17 contacts the middle region and Rpl39 the exit region.","method":"UV cross-linking of ribosome-bound nascent chains, FLAG exposure assay for nascent chain secondary structure, antibody-based identification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical cross-linking with multiple orthogonal methods distinguishing tunnel proteins","pmids":["24072706"],"is_preprint":false},{"year":1998,"finding":"Yeast Rpl4 binds specifically to the 35S precursor rRNA in vitro (Ka = 4.4×10⁶/M) but not to mature 25S rRNA, indicating L4 is one of the earliest ribosomal proteins to engage the pre-rRNA during 60S biogenesis.","method":"Modified membrane filtration assay and agarose gel mobility shift assay with in vitro synthesized 35S pre-rRNA and purified ribosomal proteins","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro binding reconstitution, single lab, no mutagenesis to confirm specificity","pmids":["9838082"],"is_preprint":false},{"year":2016,"finding":"Human RPL4 directly interacts with the central acidic domain of MDM2, suppresses MDM2-mediated p53 ubiquitination and degradation, leading to p53 stabilization and activation; RPL4 overexpression promotes MDM2 binding to RPL5 and RPL11 and formation of an RPL4–RPL5–RPL11–MDM2 complex; RPL4 knockdown also induces p53 in an RPL5/RPL11-dependent manner, indicating ribosomal stress.","method":"Co-immunoprecipitation, ubiquitination assays, Western blot for p53 stability, cell cycle analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reciprocal co-IP and functional ubiquitination assay, single lab","pmids":["26908445"],"is_preprint":false},{"year":2016,"finding":"Human RPL4 is essential for EBV Nuclear Antigen 1 (EBNA1) function: EBNA1 binds RPL4; RPL4 knockdown reduces EBNA1 activation of oriP, EBNA1 DNA binding, and EBV genome maintenance. EBV infection redistributes RPL4 to cell nuclei. RPL4 and Nucleolin form a scaffold for an EBNA1-oriP complex.","method":"Co-immunoprecipitation, shRNA knockdown, oriP luciferase reporter assay, chromatin immunoprecipitation, EBV genome copy number measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, ChIP, functional reporter, localization), single lab","pmids":["26858444"],"is_preprint":false},{"year":2005,"finding":"Human RPL4 physically interacts with RNA helicase II/Guα; the ATPase activity of Guα is required for this interaction; RPL4 knockdown inhibits 47/45S, 32S, 28S, and 18S rRNA production, and this inhibition is rescued by wild-type but not Guα-interaction-deficient RPL4, placing RPL4 as a functional partner of Guα in rRNA processing.","method":"Co-immunoprecipitation in mammalian cells, siRNA knockdown, rRNA processing assays, rescue with wild-type vs. mutant RPL4","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus siRNA knockdown with domain-mutant rescue, single lab","pmids":["16045751"],"is_preprint":false},{"year":2000,"finding":"Translational control of RPL4 mRNA is required for rapid transcription-independent neurite regeneration in PC12 cells and conditioned sensory neurons; antisense oligonucleotides to RPL4 mRNA inhibit neurite regeneration from differentiated PC12 cells and axonal elongation from conditioned sensory neurons.","method":"Subtractive hybridization to isolate RPL4 mRNA; antisense oligonucleotide inhibition; neurite regeneration assays in PC12 and sensory neurons","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — antisense loss-of-function with defined phenotypic readout, single lab","pmids":["10964612"],"is_preprint":false},{"year":1998,"finding":"La autoantigen and CNBP bind mutually exclusively to the 5′UTR of Xenopus RPL4 mRNA, assisted by Ro60 autoantigen as an ancillary factor; CNBP binds as a dimer; mutations in the 5′UTR that disrupt translational control in vivo also disrupt protein binding, implicating these interactions in translational regulation of RPL4.","method":"In vitro RNA–protein binding assays, mutational analysis of 5′UTR, competition assays, antibody perturbation, in vivo translational control assays","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal binding methods with in vivo corroboration, single lab","pmids":["9710533"],"is_preprint":false},{"year":1993,"finding":"Human RPL4 encodes a 425-amino acid protein (47,821 Da) with a 5′UTR initiating with 12 pyrimidines characteristic of vertebrate ribosomal protein mRNAs; comparison across species reveals a conserved N-terminus and a divergent C-terminus.","method":"cDNA cloning and sequencing; deduced amino acid sequence and comparison","journal":"Biochimica et biophysica acta","confidence":"Low","confidence_rationale":"Tier 4 / Weak — sequence/cloning study, no direct functional experiment","pmids":["8268230"],"is_preprint":false},{"year":2015,"finding":"RPL4 interacts with viral protein VP3 of infectious bursal disease virus (IBDV); RPL4 knockdown reduces viral protein pVP2 expression and virus titers in DF1 cells, indicating RPL4 supports IBDV replication.","method":"Immunoprecipitation–mass spectrometry screening, co-immunoprecipitation, confocal colocalization, siRNA knockdown","journal":"Virus research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP validated interaction with loss-of-function phenotype, single lab","pmids":["26415754"],"is_preprint":false},{"year":2022,"finding":"TTC22 directly interacts with RPL4 and promotes binding of WTAP mRNA to RPL4, enhancing WTAP mRNA stability and translation; RPL4 knockdown diminishes TTC22-induced increases in m6A modification levels and downstream SNAI1 upregulation.","method":"Co-immunoprecipitation, mRNA stability assays, siRNA knockdown, m6A quantification","journal":"Oncogene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, co-IP and mRNA stability, no structural or in vitro reconstitution","pmids":["35798874"],"is_preprint":false},{"year":1999,"finding":"L4 mutations in S. pneumoniae (G69C substitution; SQ insertion between Q67 and K68 in the conserved KPWRQKGTGRAR motif) confer macrolide resistance in strains without known resistance determinants, establishing that L4 loop mutations can cause macrolide resistance in a pathogen.","method":"Serial passage selection, sequencing of 23S rRNA and L4 genes, MIC determination","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 2 / Strong — replicated across multiple organisms, mechanistic basis established by structural analogy with ribosome exit tunnel","pmids":["10898684"],"is_preprint":false},{"year":2005,"finding":"6-bp deletions in the L4 ribosomal protein gene of S. pneumoniae confer resistance to macrolides and chloramphenicol and nonsusceptibility to linezolid; gene transformation of susceptible strain R6 confirmed causality, with a fitness cost observed.","method":"Sequencing, gene transformation/complementation, MIC determination","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — causality established by gene transformation, replicated findings consistent with multiple prior reports","pmids":["16048983"],"is_preprint":false}],"current_model":"RPL4 (uL4) is a universally conserved 50S/60S ribosomal protein that directly contacts 23S/28S rRNA via a long internal loop (cross-linked at Tyr35–U615 in E. coli) and a eukaryote-specific surface extension; in bacteria it autogenously represses the S10 operon at both the transcriptional level (by stabilizing a NusA-dependent RNA polymerase pause at an attenuator hairpin via direct mRNA leader binding) and the translational level, using structurally distinct RNA-binding surfaces for rRNA versus mRNA; it also inhibits RNase E activity to broadly stabilize stress-response mRNAs; in eukaryotes, newly synthesized RPL4 is co-translationally captured by the dedicated chaperone Acl4 (which sequesters the internal loop and protects it from degradation via overlapping Kap104 binding), escorted to the nucleus, and hierarchically incorporated into pre-60S particles through contacts of its eukaryote-specific extension with neighboring RpL18; within the assembled ribosome, RPL4 lines the peptide exit tunnel and contacts nascent chains throughout its length; mutations in the conserved loop tip confer macrolide (and, in some organisms, linezolid/chloramphenicol) resistance by perturbing 23S rRNA domain II/V conformation; extraribosomally, human RPL4 binds MDM2's acidic domain to suppress p53 ubiquitination and cooperates with Nucleolin to support EBNA1-mediated EBV episome maintenance."},"narrative":{"mechanistic_narrative":"RPL4 (uL4) is a universally conserved large-ribosomal-subunit protein that makes direct, extended contacts with the large-subunit rRNA and is among the earliest proteins to engage the pre-rRNA during subunit biogenesis [PMID:814400, PMID:9838082]. In the assembled ribosome it contacts the 23S/large-subunit rRNA at defined positions (Tyr35–U615 and nucleotides 320–325 in E. coli) and lines the peptide exit tunnel, uniquely contacting nascent chains along the entire tunnel length [PMID:6998491, PMID:3278299, PMID:24072706]. A conserved internal loop is critical for late assembly steps, while full-length depletion blocks early assembly, defining two temporally separable biogenesis roles [PMID:25246649]; in eukaryotes the nascent protein is co-translationally captured by the dedicated chaperone Acl4, whose TPR domain sequesters the exposed internal loop to prevent premature rRNA insertion and whose binding site overlaps that of the transport factor Kap104 to provide continuous protection during nuclear escort, with a eukaryote-specific extension contacting Rpl18 to orchestrate chaperone release and pre-60S incorporation [PMID:25936803, PMID:26447800, PMID:28148929]. Beyond its structural role, bacterial L4 is the autogenous regulator of its own S10 operon, acting both translationally and by stabilizing a NusA-dependent transcriptional pause at an attenuator hairpin through direct binding to the mRNA leader using an RNA-binding surface structurally distinct from its rRNA-binding interface [PMID:6996835, PMID:6157482, PMID:2157208, PMID:12738792, PMID:8846294]; L4 also binds and inhibits RNase E to broadly stabilize stress-response transcripts [PMID:19144914]. Mutations in the conserved loop tip confer macrolide (and in some organisms chloramphenicol/linezolid) resistance by perturbing higher-order 23S rRNA conformation in domains II, III and V and slowing peptide-bond formation and elongation [PMID:10369764, PMID:17956547, PMID:10898684, PMID:16048983]. In humans, RPL4 acts extraribosomally by binding the MDM2 acidic domain to suppress p53 ubiquitination and promote an RPL4–RPL5–RPL11–MDM2 complex [PMID:26908445], and supports rRNA processing through interaction with the RNA helicase Guα [PMID:16045751].","teleology":[{"year":1975,"claim":"Establishing that L4 directly touches rRNA in the assembled ribosome anchored its structural role within the large subunit.","evidence":"UV cross-linking of E. coli 50S subunits with immunological identification","pmids":["814400"],"confidence":"High","gaps":["Did not map the nucleotide contact site","Did not address function of the contact"]},{"year":1980,"claim":"Mapping the cross-link to Tyr35 of L4 and U615 of 23S rRNA pinpointed the precise rRNA contact and demonstrated the molecular intimacy of the L4–rRNA interface.","evidence":"Protease/nuclease digestion of isolated cross-linked complexes with oligonucleotide analysis","pmids":["6998491"],"confidence":"High","gaps":["Single contact point identified","Functional consequence of this contact not tested"]},{"year":1980,"claim":"Showing that L4 alone among S10 operon proteins represses translation of the operon, both in vitro and on overexpression in vivo, identified L4 as a self-limiting feedback regulator of ribosomal protein synthesis.","evidence":"DNA-directed in vitro synthesis with purified L4 plus in vivo oversynthesis assays","pmids":["6996835","6157482"],"confidence":"High","gaps":["Mechanism of translational inhibition not resolved","mRNA target site not defined"]},{"year":1990,"claim":"Discovery that L4 also stimulates premature transcription termination in the S10 leader, independent of translational control, revealed a second, transcriptional layer of autogenous regulation.","evidence":"In vivo RNA end-mapping plus reconstituted in vitro transcription with purified L4 and NusA","pmids":["1692593","2157208"],"confidence":"High","gaps":["How L4 stabilizes the paused complex left for later work","Did not define leader RNA elements involved"]},{"year":1995,"claim":"Dissecting the attenuator showed NusA establishes the polymerase pause while distinct leader elements allow L4 to stabilize it, separating the pause and stabilization functions.","evidence":"Genetic and antisense-oligonucleotide dissection of a purified in vitro transcription system","pmids":["7844821","7531246","1285127"],"confidence":"High","gaps":["Atomic structure of the L4-NusA-RNAP complex not determined"]},{"year":1996,"claim":"Isolation of mutants that separate ribosome assembly from autogenous control proved L4 uses non-identical RNA-binding surfaces for rRNA versus mRNA recognition.","evidence":"Genetic isolation of L4 mutants with orthogonal assembly and regulation readouts","pmids":["8846294"],"confidence":"High","gaps":["Structural basis of the two interfaces awaited crystallography"]},{"year":1999,"claim":"Demonstrating direct, specific L4 binding to a hairpin in the S10 mRNA leader, with three-dimensional mimicry of the rRNA site, explained how one protein recognizes both RNAs.","evidence":"In vitro binding, competition with the rRNA site, and phosphorothioate footprinting","pmids":["12738792"],"confidence":"High","gaps":["Co-structure of L4 with mRNA leader not solved"]},{"year":1999,"claim":"Showing L4 erythromycin-resistance mutations perturb 23S rRNA domains II/III/V and lower peptidyl-transferase activity established that L4 mutations act allosterically through rRNA conformation rather than direct drug contact.","evidence":"Chemical probing of 23S rRNA in defined resistance-mutant ribosomes","pmids":["10369764"],"confidence":"High","gaps":["Did not resolve the resistant ribosome structure","Link to nascent-chain handling not addressed"]},{"year":2000,"claim":"A 1.7 Å crystal structure of L4 provided an atomic framework, identifying separate N-terminal rRNA and C-terminal mRNA/protein binding sites and a large disordered loop.","evidence":"X-ray crystallography of Thermotoga maritima L4","pmids":["10698923"],"confidence":"High","gaps":["Loop ordered only in context of RNA/chaperone","Eukaryotic extension not represented"]},{"year":2005,"claim":"Identifying physical and functional partnership with RNA helicase Guα extended RPL4's role into eukaryotic rRNA processing.","evidence":"Co-IP, siRNA knockdown, and rescue with interaction-deficient RPL4 in mammalian cells","pmids":["16045751"],"confidence":"Medium","gaps":["Single lab","Direct enzymatic mechanism on rRNA not reconstituted"]},{"year":2009,"claim":"Discovery that L4 binds and inhibits RNase E and broadly stabilizes stress-response mRNAs revealed an extraribosomal moonlighting role in RNA turnover.","evidence":"Affinity purification, two-hybrid, in vitro cleavage and in vivo mRNA half-life and microarray assays","pmids":["19144914"],"confidence":"High","gaps":["Physiological trigger linking ribosome status to RNase E inhibition not defined"]},{"year":2014,"claim":"Separating the consequences of deleting the internal loop versus depleting full L4 defined two distinct biogenesis roles and showed the eukaryote-specific extension is dispensable for viability.","evidence":"Yeast conditional depletion/deletion mutants with ribosome profiling and pulse-chase","pmids":["25246649"],"confidence":"High","gaps":["Molecular events of early versus late assembly steps not resolved at structural level"]},{"year":2015,"claim":"Identification of the dedicated chaperone Acl4, which sequesters the internal loop and is escorted to the nucleus, explained how the aggregation-prone nascent L4 is protected and hierarchically incorporated via an Rpl18 co-evolved site.","evidence":"Yeast genetics, co-IP, co-translational capture assays and mutagenesis of contact surfaces","pmids":["25936803","26447800"],"confidence":"High","gaps":["Order of chaperone release relative to nuclear import not fully resolved"]},{"year":2016,"claim":"Demonstrating RPL4 binds the MDM2 acidic domain to suppress p53 ubiquitination connected RPL4 to the ribosomal stress–p53 surveillance axis.","evidence":"Reciprocal Co-IP, ubiquitination and p53 stability assays in human cells","pmids":["26908445"],"confidence":"Medium","gaps":["Single lab","Structural basis of MDM2 binding not defined","In vivo relevance to tumor suppression not tested"]},{"year":2016,"claim":"Finding that RPL4 cooperates with Nucleolin to support EBNA1-mediated EBV episome maintenance revealed a host-factor role hijacked by virus.","evidence":"Co-IP, shRNA knockdown, oriP reporter, ChIP and genome copy-number assays","pmids":["26858444"],"confidence":"Medium","gaps":["Single lab","Direct versus ribosome-dependent contribution not separated"]},{"year":2016,"claim":"Showing that bacterial uL4 loop deletion causes cold-sensitive assembly defects and altered response to a translation-pause peptide tied the loop to both assembly and nascent-chain sensing.","evidence":"E. coli strains expressing only loop-deleted uL4 with sedimentation and in vivo pausing assays","pmids":["27257065"],"confidence":"High","gaps":["Mechanism by which loop senses specific pause peptides not resolved"]},{"year":null,"claim":"How RPL4's extraribosomal functions (MDM2/p53, Guα-dependent processing, viral host-factor roles) are coordinated with its ribosomal duties, and whether they are conserved or context-specific, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of human RPL4 in extraribosomal complexes","Physiological signals partitioning ribosomal versus moonlighting pools unknown","No human disease link established in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[3,4,9,22]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[2,19,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,15,23]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6,7]}],"localization":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[2,19,21]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[17,24]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[22,25]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[16,17,19,21]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[22,25]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,6,7]}],"complexes":["60S/large ribosomal subunit","S10 operon attenuation complex (with NusA/RNAP)","RPL4-RPL5-RPL11-MDM2 complex","Rpl4-Acl4 chaperone complex"],"partners":["ACL4","KAP104","RPL18","NUSA","RNASE E","MDM2","EBNA1","NUCLEOLIN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P36578","full_name":"Large ribosomal subunit protein uL4","aliases":["60S ribosomal protein L1","60S ribosomal protein L4"],"length_aa":427,"mass_kda":47.7,"function":"Component of the large ribosomal subunit. 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ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA.","date":"1998","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/9838082","citation_count":13,"is_preprint":false},{"pmid":"25954255","id":"PMC_25954255","title":"Adenoviral L4 33K forms ring-like oligomers and stimulates ATPase activity of IVa2: implications in viral genome packaging.","date":"2015","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/25954255","citation_count":13,"is_preprint":false},{"pmid":"35457153","id":"PMC_35457153","title":"Tandem Mass Tagging (TMT) Reveals Tissue-Specific Proteome of L4 Larvae of Anisakis simplex s. s.: Enzymes of Energy and/or Carbohydrate Metabolism as Potential Drug Targets in Anisakiasis.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35457153","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48205,"output_tokens":7458,"usd":0.128243,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16856,"output_tokens":4688,"usd":0.10074,"stage2_stop_reason":"end_turn"},"total_usd":0.228983,"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\": 1980,\n      \"finding\": \"E. coli ribosomal protein L4 autogenously regulates the S10 operon by inhibiting translation of the promoter-proximal proteins (S10, L3, L4, L23, L2) in vitro; no other S10 operon protein caused selective inhibition, and L4 did not inhibit other operons.\",\n      \"method\": \"Lambda fus3 DNA-directed in vitro protein synthesis system with purified L4 protein addition\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified protein, replicated in companion paper (PMID:6157482)\",\n      \"pmids\": [\"6996835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1980,\n      \"finding\": \"E. coli ribosomal protein L4, when overproduced in vivo, reduces mRNA synthesis from at least four genes of the S10 operon, identifying L4 as the sole autogenous regulator among eleven S10 operon products.\",\n      \"method\": \"In vivo oversynthesis of individual S10 operon ribosomal proteins with measurement of individual protein synthesis rates\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic overexpression, replicated alongside in vitro reconstitution (PMID:6996835)\",\n      \"pmids\": [\"6157482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1975,\n      \"finding\": \"Ribosomal protein L4 is the primary RNA-crosslinked protein in the E. coli 50S subunit upon UV irradiation, demonstrating direct RNA-protein contact in the ribosome.\",\n      \"method\": \"UV cross-linking of 50S subunits followed by Sarkosyl gel analysis, 2D electrophoresis, and immunological identification with protein-specific antisera\",\n      \"journal\": \"Molecular & general genetics : MGG\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical cross-linking with multiple orthogonal verification methods\",\n      \"pmids\": [\"814400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1980,\n      \"finding\": \"The UV cross-link between L4 and 23S rRNA is located at tyrosine-35 of L4 and uridine-615 of 23S rRNA, establishing the precise RNA-protein contact site.\",\n      \"method\": \"Isolation of cross-linked L4-oligonucleotide complexes, successive protease and nuclease digestions, 2D gel electrophoresis, oligonucleotide analysis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — precise biochemical mapping with multiple orthogonal digestion/analysis methods\",\n      \"pmids\": [\"6998491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"L4 is cross-linked to nucleotides 320–325 of E. coli 23S rRNA when 50S subunits are treated with 2-iminothiolane followed by UV irradiation, identifying an additional RNA contact site.\",\n      \"method\": \"Chemical cross-linking (2-iminothiolane + UV), affinity chromatography with antibodies, partial RNA digestion, complex isolation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous biochemical method but single lab, single study\",\n      \"pmids\": [\"3278299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"L4 stimulates premature transcription termination approximately 140 bases from the transcription start site within the S10 operon leader, upstream of the first structural gene, by a mechanism independent of its inhibition of translation.\",\n      \"method\": \"In vivo mapping of 5′ and 3′ ends of newly synthesized RNA; deletion of sequences downstream of termination site\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo mapping plus genetic deletion to dissociate transcriptional from translational control, replicated in vitro (PMID:2157208)\",\n      \"pmids\": [\"1692593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"L4 stimulates termination of transcription at the S10 attenuator in vitro, but RNA polymerase requires NusA to terminate at this site; L4 increases termination efficiency at the NusA-dependent pause site.\",\n      \"method\": \"Purified in vitro transcription system with purified L4 and NusA proteins\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro transcription with purified components, replicated across multiple papers\",\n      \"pmids\": [\"2157208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"NusA is required for RNA polymerase pausing at the S10 attenuation site; L4 further stabilizes the NusA-modified paused complex. These two activities require genetically separable regions of the S10 leader RNA.\",\n      \"method\": \"In vitro transcription with purified components; isolation of paused complexes; genetic deletion analysis of leader RNA\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro with purified proteins plus genetic dissection\",\n      \"pmids\": [\"1285127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The upper stem-loop of the attenuator hairpin is the major determinant for NusA-dependent pausing; upstream hairpin and the ascending side of the attenuator are required for L4 stabilization of the paused complex; NusA is not absolutely required for initial RNA polymerase pausing at low UTP concentration, but is required for L4 to stabilize the pause.\",\n      \"method\": \"Genetic and antisense oligonucleotide competition with purified in vitro transcription system; isolation of paused complexes\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution combined with genetic and oligonucleotide-based dissection\",\n      \"pmids\": [\"7844821\", \"7531246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"E. coli L4 binds directly and specifically to the S10 mRNA leader in vitro; the binding site is a small hairpin structure within the leader but a 64-nucleotide sequence is required for full interaction. Phosphorothioate footprinting shows structural mimicry between the mRNA and rRNA binding sites in three dimensions.\",\n      \"method\": \"In vitro binding assays, competition with 23S rRNA L4-binding site, phosphorothioate footprinting, mutational analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro binding reconstitution with footprinting and mutagenesis in one study\",\n      \"pmids\": [\"12738792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"L4 uses non-identical determinants for ribosome assembly and autogenous S10 operon regulation: mutations were isolated that eliminate autogenous control but allow ribosome assembly, and vice versa, showing the two RNA-binding interfaces are distinct.\",\n      \"method\": \"Genetic isolation of L4 mutants; functional assays for ribosome assembly and autogenous control\",\n      \"journal\": \"RNA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic dissection with multiple separable mutants, two orthogonal functional readouts\",\n      \"pmids\": [\"8846294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of Thermotoga maritima L4 at 1.7 Å resolution reveals an alternating α/β fold with a large disordered loop; two separate RNA-binding sites are identified: an N-terminal site (disordered loop with flanking helices) for rRNA binding and a C-terminal site (two non-consecutive helices) candidate for S10 mRNA leader interaction; a C-terminal protein-binding interface is also identified.\",\n      \"method\": \"X-ray crystallography at 1.7 Å resolution\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mapping of binding sites\",\n      \"pmids\": [\"10698923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Erythromycin resistance mutations in ribosomal protein L4 (but not L22) of E. coli result in decreased peptide-bond-forming activity and perturb the conformation of 23S rRNA nucleotides in domains II, III, and V, without affecting the A2058 region—indicating L4 mutations act by altering rRNA higher-order structure.\",\n      \"method\": \"Chemical modification/probing of 23S rRNA structure in ribosomes from resistance mutants; multiple chemical probes at multiple nucleotide positions\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct structural probing with multiple reagents on defined mutant ribosomes\",\n      \"pmids\": [\"10369764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"L4 mutant ribosomes with altered L4 protein show increased frameshifting, missense decoding, stop codon readthrough, non-AUG initiation, and mutant initiator tRNA utilization, demonstrating that L4 mutations in the 50S subunit also alter 30S subunit structure and function upon association.\",\n      \"method\": \"In vivo translational fidelity assays (frameshifting, readthrough, missense) in E. coli L4 and L22 mutant strains; antibiotic interaction assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vivo functional assays in defined mutant strains\",\n      \"pmids\": [\"15509870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Eight new L4 mutations in the tentacle region of E. coli L4 confer erythromycin resistance; all reduce in vivo peptide-chain elongation rates and increase precursor 23S rRNA levels; large insertions in L4 cause accumulation of abnormal ribosomal subunits, highlighting L4's role in ribosome assembly and function.\",\n      \"method\": \"In vivo isolation of erythromycin-resistant mutants; sequencing; growth rate measurements; ribosome profiling; rRNA precursor analysis\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple mutants characterized with multiple orthogonal functional readouts\",\n      \"pmids\": [\"17956547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"E. coli ribosomal protein L4 interacts with RNase E outside its catalytic domain and inhibits RNase E endoribonucleolytic activity in vitro; in vivo, ectopic L4 stabilizes RNase E-targeted mRNAs and stabilizes an antisense regulatory RNA controlling plasmid replication; L4 overexpression broadens mRNA stability for stress-response transcripts.\",\n      \"method\": \"Affinity purification, immunoprecipitation, E. coli two-hybrid screening, in vitro cleavage assays, mRNA half-life measurements, DNA microarray\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including in vitro reconstitution and in vivo mRNA stability assays\",\n      \"pmids\": [\"19144914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Eukaryotic Rpl4 (yeast) requires a dedicated assembly chaperone Acl4, which binds the universally conserved internal loop of newly synthesized Rpl4 via a superhelical TPR domain, preventing premature rRNA insertion. Rpl4's eukaryote-specific extension makes distinct interactions with the 60S surface (including a co-evolved site on Rpl18) that orchestrate Acl4 release and Rpl4 pre-ribosome incorporation.\",\n      \"method\": \"Yeast genetics, biochemical pulldowns, co-IP, mutational analysis of RpL4 and RpL18 contact sites, ribosome assembly assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetics, biochemistry, mutagenesis of interacting surfaces) in a single rigorous study\",\n      \"pmids\": [\"25936803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Acl4 acts as dedicated chaperone that binds the long internal loop of Rpl4 (yeast), accompanies it from the cytoplasm to the nucleus, and is required for 60S subunit production; both the internal loop and C-terminal eukaryote-specific extension are essential for Rpl4 function; Rpl4 contains at least five nuclear localization signals.\",\n      \"method\": \"Yeast genetics (slow-growth and 60S deficiency phenotypes), biochemical co-fractionation, co-translational capture assays, localization studies\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic and biochemical methods replicating findings of PMID:25936803\",\n      \"pmids\": [\"26447800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of yeast Rpl4 bound to its chaperone Acl4 reveals extensive interactions sequestering 70 exposed residues of the extended Rpl4 loop; the eukaryote-specific Rpl4 extension harbors overlapping binding sites for Acl4 and nuclear transport factor Kap104, enabling continuous protection from cellular degradation machinery.\",\n      \"method\": \"X-ray crystallography of Rpl4–Acl4 complex; biochemical binding assays for Kap104 competition; degradation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus biochemical validation of overlapping binding sites\",\n      \"pmids\": [\"28148929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In yeast, deletion of Rpl4's conserved internal loop impairs growth and reduces large ribosomal subunit levels, affecting later steps in assembly; depletion of full Rpl4 blocks early assembly steps, revealing two distinct roles for L4 in ribosome biogenesis. Deletion of the entire eukaryote-specific C-terminal extension has no effect on viability or 60S production.\",\n      \"method\": \"Yeast genetics (conditional depletion, deletion mutants), ribosome profiling, polysome analysis, pulse-chase labeling\",\n      \"journal\": \"RNA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple deletion mutants with defined assembly phenotypes and orthogonal ribosome analyses\",\n      \"pmids\": [\"25246649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In E. coli, deletion of the uL4 loop causes cold-sensitive ribosome assembly defects and accumulation of immature particles; the uL4 loop deletion also impairs response to the cmlA(crb) translation pause peptide but not to the secM pause peptide, while uL22 loop deletion affects different assembly intermediates.\",\n      \"method\": \"Genetic analysis using strains expressing only loop-deleted uL4; ribosome sedimentation, rRNA processing analysis, in vivo pausing assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — defined genetic strains with multiple orthogonal assays for assembly and translation function\",\n      \"pmids\": [\"27257065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In yeast, ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 all contact the signal anchor of nascent chains within the exit tunnel; Rpl4 uniquely contacts nascent chain residues throughout the entire ribosomal tunnel length, whereas Rpl17 contacts the middle region and Rpl39 the exit region.\",\n      \"method\": \"UV cross-linking of ribosome-bound nascent chains, FLAG exposure assay for nascent chain secondary structure, antibody-based identification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical cross-linking with multiple orthogonal methods distinguishing tunnel proteins\",\n      \"pmids\": [\"24072706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Yeast Rpl4 binds specifically to the 35S precursor rRNA in vitro (Ka = 4.4×10⁶/M) but not to mature 25S rRNA, indicating L4 is one of the earliest ribosomal proteins to engage the pre-rRNA during 60S biogenesis.\",\n      \"method\": \"Modified membrane filtration assay and agarose gel mobility shift assay with in vitro synthesized 35S pre-rRNA and purified ribosomal proteins\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro binding reconstitution, single lab, no mutagenesis to confirm specificity\",\n      \"pmids\": [\"9838082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Human RPL4 directly interacts with the central acidic domain of MDM2, suppresses MDM2-mediated p53 ubiquitination and degradation, leading to p53 stabilization and activation; RPL4 overexpression promotes MDM2 binding to RPL5 and RPL11 and formation of an RPL4–RPL5–RPL11–MDM2 complex; RPL4 knockdown also induces p53 in an RPL5/RPL11-dependent manner, indicating ribosomal stress.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, Western blot for p53 stability, cell cycle analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reciprocal co-IP and functional ubiquitination assay, single lab\",\n      \"pmids\": [\"26908445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Human RPL4 is essential for EBV Nuclear Antigen 1 (EBNA1) function: EBNA1 binds RPL4; RPL4 knockdown reduces EBNA1 activation of oriP, EBNA1 DNA binding, and EBV genome maintenance. EBV infection redistributes RPL4 to cell nuclei. RPL4 and Nucleolin form a scaffold for an EBNA1-oriP complex.\",\n      \"method\": \"Co-immunoprecipitation, shRNA knockdown, oriP luciferase reporter assay, chromatin immunoprecipitation, EBV genome copy number measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, ChIP, functional reporter, localization), single lab\",\n      \"pmids\": [\"26858444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human RPL4 physically interacts with RNA helicase II/Guα; the ATPase activity of Guα is required for this interaction; RPL4 knockdown inhibits 47/45S, 32S, 28S, and 18S rRNA production, and this inhibition is rescued by wild-type but not Guα-interaction-deficient RPL4, placing RPL4 as a functional partner of Guα in rRNA processing.\",\n      \"method\": \"Co-immunoprecipitation in mammalian cells, siRNA knockdown, rRNA processing assays, rescue with wild-type vs. mutant RPL4\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus siRNA knockdown with domain-mutant rescue, single lab\",\n      \"pmids\": [\"16045751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Translational control of RPL4 mRNA is required for rapid transcription-independent neurite regeneration in PC12 cells and conditioned sensory neurons; antisense oligonucleotides to RPL4 mRNA inhibit neurite regeneration from differentiated PC12 cells and axonal elongation from conditioned sensory neurons.\",\n      \"method\": \"Subtractive hybridization to isolate RPL4 mRNA; antisense oligonucleotide inhibition; neurite regeneration assays in PC12 and sensory neurons\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — antisense loss-of-function with defined phenotypic readout, single lab\",\n      \"pmids\": [\"10964612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"La autoantigen and CNBP bind mutually exclusively to the 5′UTR of Xenopus RPL4 mRNA, assisted by Ro60 autoantigen as an ancillary factor; CNBP binds as a dimer; mutations in the 5′UTR that disrupt translational control in vivo also disrupt protein binding, implicating these interactions in translational regulation of RPL4.\",\n      \"method\": \"In vitro RNA–protein binding assays, mutational analysis of 5′UTR, competition assays, antibody perturbation, in vivo translational control assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal binding methods with in vivo corroboration, single lab\",\n      \"pmids\": [\"9710533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Human RPL4 encodes a 425-amino acid protein (47,821 Da) with a 5′UTR initiating with 12 pyrimidines characteristic of vertebrate ribosomal protein mRNAs; comparison across species reveals a conserved N-terminus and a divergent C-terminus.\",\n      \"method\": \"cDNA cloning and sequencing; deduced amino acid sequence and comparison\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — sequence/cloning study, no direct functional experiment\",\n      \"pmids\": [\"8268230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RPL4 interacts with viral protein VP3 of infectious bursal disease virus (IBDV); RPL4 knockdown reduces viral protein pVP2 expression and virus titers in DF1 cells, indicating RPL4 supports IBDV replication.\",\n      \"method\": \"Immunoprecipitation–mass spectrometry screening, co-immunoprecipitation, confocal colocalization, siRNA knockdown\",\n      \"journal\": \"Virus research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP validated interaction with loss-of-function phenotype, single lab\",\n      \"pmids\": [\"26415754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TTC22 directly interacts with RPL4 and promotes binding of WTAP mRNA to RPL4, enhancing WTAP mRNA stability and translation; RPL4 knockdown diminishes TTC22-induced increases in m6A modification levels and downstream SNAI1 upregulation.\",\n      \"method\": \"Co-immunoprecipitation, mRNA stability assays, siRNA knockdown, m6A quantification\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, co-IP and mRNA stability, no structural or in vitro reconstitution\",\n      \"pmids\": [\"35798874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"L4 mutations in S. pneumoniae (G69C substitution; SQ insertion between Q67 and K68 in the conserved KPWRQKGTGRAR motif) confer macrolide resistance in strains without known resistance determinants, establishing that L4 loop mutations can cause macrolide resistance in a pathogen.\",\n      \"method\": \"Serial passage selection, sequencing of 23S rRNA and L4 genes, MIC determination\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Strong — replicated across multiple organisms, mechanistic basis established by structural analogy with ribosome exit tunnel\",\n      \"pmids\": [\"10898684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"6-bp deletions in the L4 ribosomal protein gene of S. pneumoniae confer resistance to macrolides and chloramphenicol and nonsusceptibility to linezolid; gene transformation of susceptible strain R6 confirmed causality, with a fitness cost observed.\",\n      \"method\": \"Sequencing, gene transformation/complementation, MIC determination\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — causality established by gene transformation, replicated findings consistent with multiple prior reports\",\n      \"pmids\": [\"16048983\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RPL4 (uL4) is a universally conserved 50S/60S ribosomal protein that directly contacts 23S/28S rRNA via a long internal loop (cross-linked at Tyr35–U615 in E. coli) and a eukaryote-specific surface extension; in bacteria it autogenously represses the S10 operon at both the transcriptional level (by stabilizing a NusA-dependent RNA polymerase pause at an attenuator hairpin via direct mRNA leader binding) and the translational level, using structurally distinct RNA-binding surfaces for rRNA versus mRNA; it also inhibits RNase E activity to broadly stabilize stress-response mRNAs; in eukaryotes, newly synthesized RPL4 is co-translationally captured by the dedicated chaperone Acl4 (which sequesters the internal loop and protects it from degradation via overlapping Kap104 binding), escorted to the nucleus, and hierarchically incorporated into pre-60S particles through contacts of its eukaryote-specific extension with neighboring RpL18; within the assembled ribosome, RPL4 lines the peptide exit tunnel and contacts nascent chains throughout its length; mutations in the conserved loop tip confer macrolide (and, in some organisms, linezolid/chloramphenicol) resistance by perturbing 23S rRNA domain II/V conformation; extraribosomally, human RPL4 binds MDM2's acidic domain to suppress p53 ubiquitination and cooperates with Nucleolin to support EBNA1-mediated EBV episome maintenance.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RPL4 (uL4) is a universally conserved large-ribosomal-subunit protein that makes direct, extended contacts with the large-subunit rRNA and is among the earliest proteins to engage the pre-rRNA during subunit biogenesis [#2, #22]. In the assembled ribosome it contacts the 23S/large-subunit rRNA at defined positions (Tyr35–U615 and nucleotides 320–325 in E. coli) and lines the peptide exit tunnel, uniquely contacting nascent chains along the entire tunnel length [#3, #4, #21]. A conserved internal loop is critical for late assembly steps, while full-length depletion blocks early assembly, defining two temporally separable biogenesis roles [#19]; in eukaryotes the nascent protein is co-translationally captured by the dedicated chaperone Acl4, whose TPR domain sequesters the exposed internal loop to prevent premature rRNA insertion and whose binding site overlaps that of the transport factor Kap104 to provide continuous protection during nuclear escort, with a eukaryote-specific extension contacting Rpl18 to orchestrate chaperone release and pre-60S incorporation [#16, #17, #18]. Beyond its structural role, bacterial L4 is the autogenous regulator of its own S10 operon, acting both translationally and by stabilizing a NusA-dependent transcriptional pause at an attenuator hairpin through direct binding to the mRNA leader using an RNA-binding surface structurally distinct from its rRNA-binding interface [#0, #1, #6, #9, #10]; L4 also binds and inhibits RNase E to broadly stabilize stress-response transcripts [#15]. Mutations in the conserved loop tip confer macrolide (and in some organisms chloramphenicol/linezolid) resistance by perturbing higher-order 23S rRNA conformation in domains II, III and V and slowing peptide-bond formation and elongation [#12, #14, #31, #32]. In humans, RPL4 acts extraribosomally by binding the MDM2 acidic domain to suppress p53 ubiquitination and promote an RPL4–RPL5–RPL11–MDM2 complex [#23], and supports rRNA processing through interaction with the RNA helicase Guα [#25].\",\n  \"teleology\": [\n    {\n      \"year\": 1975,\n      \"claim\": \"Establishing that L4 directly touches rRNA in the assembled ribosome anchored its structural role within the large subunit.\",\n      \"evidence\": \"UV cross-linking of E. coli 50S subunits with immunological identification\",\n      \"pmids\": [\"814400\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the nucleotide contact site\", \"Did not address function of the contact\"]\n    },\n    {\n      \"year\": 1980,\n      \"claim\": \"Mapping the cross-link to Tyr35 of L4 and U615 of 23S rRNA pinpointed the precise rRNA contact and demonstrated the molecular intimacy of the L4–rRNA interface.\",\n      \"evidence\": \"Protease/nuclease digestion of isolated cross-linked complexes with oligonucleotide analysis\",\n      \"pmids\": [\"6998491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single contact point identified\", \"Functional consequence of this contact not tested\"]\n    },\n    {\n      \"year\": 1980,\n      \"claim\": \"Showing that L4 alone among S10 operon proteins represses translation of the operon, both in vitro and on overexpression in vivo, identified L4 as a self-limiting feedback regulator of ribosomal protein synthesis.\",\n      \"evidence\": \"DNA-directed in vitro synthesis with purified L4 plus in vivo oversynthesis assays\",\n      \"pmids\": [\"6996835\", \"6157482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of translational inhibition not resolved\", \"mRNA target site not defined\"]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Discovery that L4 also stimulates premature transcription termination in the S10 leader, independent of translational control, revealed a second, transcriptional layer of autogenous regulation.\",\n      \"evidence\": \"In vivo RNA end-mapping plus reconstituted in vitro transcription with purified L4 and NusA\",\n      \"pmids\": [\"1692593\", \"2157208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How L4 stabilizes the paused complex left for later work\", \"Did not define leader RNA elements involved\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Dissecting the attenuator showed NusA establishes the polymerase pause while distinct leader elements allow L4 to stabilize it, separating the pause and stabilization functions.\",\n      \"evidence\": \"Genetic and antisense-oligonucleotide dissection of a purified in vitro transcription system\",\n      \"pmids\": [\"7844821\", \"7531246\", \"1285127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the L4-NusA-RNAP complex not determined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Isolation of mutants that separate ribosome assembly from autogenous control proved L4 uses non-identical RNA-binding surfaces for rRNA versus mRNA recognition.\",\n      \"evidence\": \"Genetic isolation of L4 mutants with orthogonal assembly and regulation readouts\",\n      \"pmids\": [\"8846294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the two interfaces awaited crystallography\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrating direct, specific L4 binding to a hairpin in the S10 mRNA leader, with three-dimensional mimicry of the rRNA site, explained how one protein recognizes both RNAs.\",\n      \"evidence\": \"In vitro binding, competition with the rRNA site, and phosphorothioate footprinting\",\n      \"pmids\": [\"12738792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Co-structure of L4 with mRNA leader not solved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Showing L4 erythromycin-resistance mutations perturb 23S rRNA domains II/III/V and lower peptidyl-transferase activity established that L4 mutations act allosterically through rRNA conformation rather than direct drug contact.\",\n      \"evidence\": \"Chemical probing of 23S rRNA in defined resistance-mutant ribosomes\",\n      \"pmids\": [\"10369764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the resistant ribosome structure\", \"Link to nascent-chain handling not addressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"A 1.7 Å crystal structure of L4 provided an atomic framework, identifying separate N-terminal rRNA and C-terminal mRNA/protein binding sites and a large disordered loop.\",\n      \"evidence\": \"X-ray crystallography of Thermotoga maritima L4\",\n      \"pmids\": [\"10698923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Loop ordered only in context of RNA/chaperone\", \"Eukaryotic extension not represented\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying physical and functional partnership with RNA helicase Guα extended RPL4's role into eukaryotic rRNA processing.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, and rescue with interaction-deficient RPL4 in mammalian cells\",\n      \"pmids\": [\"16045751\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct enzymatic mechanism on rRNA not reconstituted\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovery that L4 binds and inhibits RNase E and broadly stabilizes stress-response mRNAs revealed an extraribosomal moonlighting role in RNA turnover.\",\n      \"evidence\": \"Affinity purification, two-hybrid, in vitro cleavage and in vivo mRNA half-life and microarray assays\",\n      \"pmids\": [\"19144914\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger linking ribosome status to RNase E inhibition not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Separating the consequences of deleting the internal loop versus depleting full L4 defined two distinct biogenesis roles and showed the eukaryote-specific extension is dispensable for viability.\",\n      \"evidence\": \"Yeast conditional depletion/deletion mutants with ribosome profiling and pulse-chase\",\n      \"pmids\": [\"25246649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular events of early versus late assembly steps not resolved at structural level\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of the dedicated chaperone Acl4, which sequesters the internal loop and is escorted to the nucleus, explained how the aggregation-prone nascent L4 is protected and hierarchically incorporated via an Rpl18 co-evolved site.\",\n      \"evidence\": \"Yeast genetics, co-IP, co-translational capture assays and mutagenesis of contact surfaces\",\n      \"pmids\": [\"25936803\", \"26447800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of chaperone release relative to nuclear import not fully resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrating RPL4 binds the MDM2 acidic domain to suppress p53 ubiquitination connected RPL4 to the ribosomal stress–p53 surveillance axis.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination and p53 stability assays in human cells\",\n      \"pmids\": [\"26908445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Structural basis of MDM2 binding not defined\", \"In vivo relevance to tumor suppression not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Finding that RPL4 cooperates with Nucleolin to support EBNA1-mediated EBV episome maintenance revealed a host-factor role hijacked by virus.\",\n      \"evidence\": \"Co-IP, shRNA knockdown, oriP reporter, ChIP and genome copy-number assays\",\n      \"pmids\": [\"26858444\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct versus ribosome-dependent contribution not separated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showing that bacterial uL4 loop deletion causes cold-sensitive assembly defects and altered response to a translation-pause peptide tied the loop to both assembly and nascent-chain sensing.\",\n      \"evidence\": \"E. coli strains expressing only loop-deleted uL4 with sedimentation and in vivo pausing assays\",\n      \"pmids\": [\"27257065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which loop senses specific pause peptides not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RPL4's extraribosomal functions (MDM2/p53, Guα-dependent processing, viral host-factor roles) are coordinated with its ribosomal duties, and whether they are conserved or context-specific, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of human RPL4 in extraribosomal complexes\", \"Physiological signals partitioning ribosomal versus moonlighting pools unknown\", \"No human disease link established in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [3, 4, 9, 22]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [2, 19, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 15, 23]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [2, 19, 21]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17, 24]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [22, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [16, 17, 19, 21]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [22, 25]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 6, 7]}\n    ],\n    \"complexes\": [\n      \"60S/large ribosomal subunit\",\n      \"S10 operon attenuation complex (with NusA/RNAP)\",\n      \"RPL4-RPL5-RPL11-MDM2 complex\",\n      \"Rpl4-Acl4 chaperone complex\"\n    ],\n    \"partners\": [\n      \"Acl4\",\n      \"Kap104\",\n      \"Rpl18\",\n      \"NusA\",\n      \"RNase E\",\n      \"MDM2\",\n      \"EBNA1\",\n      \"Nucleolin\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}