{"gene":"MIF4GD","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2007,"finding":"SLIP1 (MIF4GD) was identified as a binding partner of SLBP via yeast two-hybrid screening. Five conserved residues in a 15-amino-acid region of SLBP's N-terminal portion are required for both translation activation and SLIP1 binding; mutations in any of these residues reduce SLIP1 binding. Co-expression of SLIP1 with human SLBP in Xenopus oocytes stimulates translation of a stem-loop reporter mRNA but not a polyadenylated reporter, and siRNA-mediated knockdown of SLIP1 in HeLa cells reduces histone mRNA translation rate and cell viability.","method":"Yeast two-hybrid screen, site-directed mutagenesis, Xenopus oocyte translation assay, RNAi knockdown in HeLa cells, GFP reporter assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (two-hybrid, mutagenesis, oocyte functional assay, RNAi with defined translational phenotype) in a single study with clear functional readouts","pmids":["18025107"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of zebrafish SLIP1 bound to the translation-activation domain of SLBP was solved at 2.5 Å resolution, defining the molecular determinants of SLBP recognition. A SLIP1-binding motif (SBM) was identified in two additional proteins, eIF3g and the mRNA-export factor DBP5; the 3.25 Å crystal structure of SLIP1 bound to the DBP5 SBM was also determined. Pull-down assays confirmed SLIP1 binding to DBP5 and eIF3g. SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CTIF, suggesting SLIP1 homodimer or SLIP1-CTIF heterodimer can bridge SLBP with SBM-containing proteins in mRNA metabolism.","method":"X-ray crystallography (2.5 Å and 3.25 Å resolution structures), pull-down assays, sequence conservation analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — two independent crystal structures plus biochemical pull-down confirmation in a single rigorous study","pmids":["23804756"],"is_preprint":false},{"year":2013,"finding":"SLIP1 is a homodimer that does not bind RNA on its own. Unphosphorylated SLBP-SLIP1 forms a 2:2 high-affinity (Kd <0.9 nM) heterotetramer incapable of binding histone mRNA. Phosphorylated SLBP (phosphorylated at 23 Ser/Thr sites) has weak affinity (~3 µM) for SLIP1. Sequential binding — phosphorylated SLBP to the histone mRNA stem-loop, then SLIP1 — is required to form an active ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to a heterodimer. Alanine scanning mutagenesis mapped the SLBP-binding site on SLIP1 to near the dimer interface; a single-point mutant there abolished SLBP interaction in vitro and reduced histone mRNA abundance in vivo.","method":"Biophysical characterization (sedimentation, binding assays), baculovirus phosphorylated SLBP expression, alanine scanning mutagenesis, in vivo histone mRNA measurement","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with phosphorylated and unphosphorylated proteins, quantitative binding measurements, mutagenesis with in vivo validation, multiple orthogonal methods in one study","pmids":["23286197"],"is_preprint":false},{"year":2012,"finding":"INT6/EIF3E (an eIF3 subunit) was identified as a binding partner of MIF4GD/SLIP1 via yeast two-hybrid screen. INT6 and MIF4GD co-localize in cytoplasmic foci. siRNA knockdown of INT6 reduces S-phase histone mRNA translation, assessed by endogenous histone protein levels and luciferase reporter driven by histone stem-loop elements; INT6 overexpression has the opposite effect. INT6 interacts with both MIF4GD and SLBP, placing it in the histone mRNA translation complex.","method":"Yeast two-hybrid screen, RNAi knockdown, luciferase reporter assay, co-localization by immunofluorescence","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two-hybrid plus RNAi phenotype plus co-localization, single lab, multiple methods but no reciprocal Co-IP reported in abstract","pmids":["22532700"],"is_preprint":false},{"year":2013,"finding":"MIF4GD was identified as a novel binding partner for the CDK inhibitor p27(kip1) via yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation and GST pull-down assays. MIF4GD interaction with p27 stabilizes p27 in both the nucleus and cytoplasm by suppressing CDK2-mediated phosphorylation of p27 at Thr187. MIF4GD overexpression increases p27 levels and reduces cell proliferation; MIF4GD knockdown promotes cell cycle progression with decreased p27 levels. MIF4GD overexpression reduces colony formation and inhibits xenograft tumor growth in nude mice.","method":"Yeast two-hybrid screen, co-immunoprecipitation, GST pull-down, overexpression and knockdown in hepatocellular carcinoma cells, xenograft tumor model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and GST pull-down confirming interaction, plus loss- and gain-of-function with defined cell cycle phenotype, single lab","pmids":["24336329"],"is_preprint":false},{"year":2010,"finding":"MIF4GD/SLIP1 was identified as a candidate binding partner of supervillin (via yeast two-hybrid screen); the MIF4GD-interacting sequence co-localizes with and mis-localizes EGFP-supervillin in mammalian cells, and MIF4GD-interacting sequences mimic supervillin overexpression by inhibiting cell spreading.","method":"Yeast two-hybrid screen, co-localization of EGFP-supervillin with interacting sequences in mammalian cells, cell spreading assay","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid identification with co-localization in cells, single lab, no direct biochemical confirmation of MIF4GD-supervillin interaction reported for this pair","pmids":["20309963"],"is_preprint":false},{"year":2010,"finding":"In rat testis, MIF4GD exists in three isoforms (25, 20, and 16 kDa). The 20-kDa form is testis-specific. The 16-kDa MIF4GD uniquely co-fractionates with 40S ribosomal subunits and ribosomes in spermatogenic cells by subcellular fractionation, suggesting it functions as a translational regulator in spermiogenesis.","method":"Western blot, subcellular fractionation (ribosome sedimentation), tissue expression analysis","journal":"The Journal of reproduction and development","confidence":"Low","confidence_rationale":"Tier 3 / Weak — fractionation-based localization without functional consequence directly tested, single lab, single method","pmids":["21157122"],"is_preprint":false}],"current_model":"MIF4GD (SLIP1) is a MIF4G domain-containing homodimer that functions as a translational activator of replication-dependent histone mRNAs by binding the translation-activation domain of SLBP (stem-loop binding protein); the active complex requires sequential binding of phosphorylated SLBP to the histone mRNA stem-loop followed by SLIP1 recruitment, forming a ternary complex that bridges the 3' stem-loop to the translation initiation machinery via interactions with eIF3g and mRNA-export factor DBP5, and is further regulated by INT6/EIF3E; MIF4GD also interacts with p27(kip1) to suppress CDK2-mediated phosphorylation of p27 at Thr187, thereby stabilizing p27 and restraining cell cycle progression."},"narrative":{"mechanistic_narrative":"MIF4GD (SLIP1) is a MIF4G-domain homodimer that functions as a translational activator of replication-dependent histone mRNAs by coupling the stem-loop binding protein SLBP to the translation initiation machinery [PMID:18025107, PMID:23286197]. It was identified as an SLBP partner whose binding requires a conserved 15-residue translation-activation region in the SLBP N-terminus, and its depletion lowers histone mRNA translation and cell viability [PMID:18025107]. Activation is assembled sequentially and is phosphorylation-gated: unphosphorylated SLBP and SLIP1 form a tight 2:2 heterotetramer that cannot bind RNA, whereas phosphorylated SLBP first binds the histone mRNA stem-loop and then recruits SLIP1, forming the active ternary complex, with SLBP Thr171 phosphorylation driving heterotetramer-to-heterodimer conversion [PMID:23286197]. Crystal structures of SLIP1 bound to SLBP and to a SLIP1-binding motif (SBM) define how the homodimer simultaneously engages SLBP and SBM-containing factors eIF3g and the mRNA-export factor DBP5, bridging the histone mRNA to initiation; the homodimerization and SBM-binding residues are shared with the MIF4G domain of CTIF [PMID:23804756]. The eIF3 subunit INT6/EIF3E binds both MIF4GD and SLBP and co-localizes with MIF4GD in cytoplasmic foci, promoting S-phase histone mRNA translation [PMID:22532700]. Independently of its translational role, MIF4GD binds the CDK inhibitor p27(kip1), suppresses CDK2-mediated phosphorylation of p27 at Thr187 to stabilize p27 in nucleus and cytoplasm, and thereby restrains proliferation, reducing colony formation and xenograft tumor growth [PMID:24336329].","teleology":[{"year":2007,"claim":"Established MIF4GD/SLIP1 as a dedicated activator of histone mRNA translation by identifying it as the SLBP partner that links the SLBP translation-activation domain to a functional output.","evidence":"Yeast two-hybrid screen, SLBP mutagenesis, Xenopus oocyte stem-loop reporter translation assay, and RNAi in HeLa cells","pmids":["18025107"],"confidence":"High","gaps":["Did not define the structural basis of the SLBP-SLIP1 interface","Connection to the core initiation machinery not yet identified"]},{"year":2010,"claim":"First indications of additional partners and tissue-specific forms, hinting at roles beyond the canonical SLBP complex.","evidence":"Yeast two-hybrid with supervillin plus cell-spreading assay; Western blot and ribosome fractionation of rat testis isoforms","pmids":["20309963","21157122"],"confidence":"Low","gaps":["Supervillin interaction not confirmed biochemically and functional relevance unestablished","Testis-specific 20-kDa isoform function not tested","Ribosome co-fractionation of the 16-kDa form is correlative, no functional consequence shown"]},{"year":2012,"claim":"Placed an eIF3 subunit, INT6/EIF3E, within the histone mRNA translation complex, providing a route from SLIP1/SLBP to the initiation apparatus.","evidence":"Yeast two-hybrid, RNAi and overexpression with histone stem-loop luciferase reporter, immunofluorescence co-localization","pmids":["22532700"],"confidence":"Medium","gaps":["No reciprocal Co-IP reported confirming the MIF4GD-INT6 interaction","Mechanism by which INT6 enhances translation not resolved"]},{"year":2013,"claim":"Resolved the assembly logic and structural determinants of the activation complex, showing SLIP1 is a non-RNA-binding homodimer whose recruitment is gated by SLBP phosphorylation and that it bridges SBM-containing factors eIF3g and DBP5.","evidence":"Crystal structures of SLIP1-SLBP and SLIP1-DBP5 SBM, pull-downs, biophysical reconstitution with phosphorylated/unphosphorylated SLBP, alanine scanning with in vivo histone mRNA readout","pmids":["23804756","23286197"],"confidence":"High","gaps":["In-cell stoichiometry and dynamics of the heterotetramer-to-ternary transition not directly observed","Whether SLIP1-CTIF heterodimers form in vivo not established","Role of DBP5 in histone mRNA translation not functionally tested"]},{"year":2013,"claim":"Revealed a translation-independent role in cell cycle control through stabilization of the CDK inhibitor p27, linking MIF4GD to proliferation restraint and tumor suppression.","evidence":"Yeast two-hybrid, reciprocal Co-IP and GST pull-down, gain/loss-of-function in hepatocellular carcinoma cells, xenograft tumor model","pmids":["24336329"],"confidence":"Medium","gaps":["Mechanism by which MIF4GD suppresses CDK2-mediated p27 Thr187 phosphorylation not defined","Relationship between the translational and p27-stabilizing functions unresolved","Single lab, single tumor context"]},{"year":null,"claim":"How MIF4GD partitions between its histone mRNA translation role and its p27-stabilizing cell cycle role, and whether these activities are coordinated, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model connecting translational activation and p27 stabilization","Regulation of MIF4GD itself (modifications, localization control) uncharacterized","In vivo physiological requirement not addressed by knockout"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[],"complexes":[],"partners":["SLBP","EIF3E","EIF3G","DBP5","CDKN1B","CTIF"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"A9UHW6","full_name":"MIF4G domain-containing protein","aliases":["SLBP-interacting protein 1","hSLIP1"],"length_aa":222,"mass_kda":25.4,"function":"Functions in replication-dependent translation of histone mRNAs which differ from other eukaryotic mRNAs in that they do not end with a poly-A tail but a stem-loop. May participate in circularizing those mRNAs specifically enhancing their translation","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/A9UHW6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MIF4GD","classification":"Not Classified","n_dependent_lines":19,"n_total_lines":1208,"dependency_fraction":0.015728476821192054},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MIF4GD","total_profiled":1310},"omim":[{"mim_id":"612072","title":"MIF4G DOMAIN-CONTAINING PROTEIN; MIF4GD","url":"https://www.omim.org/entry/612072"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MIF4GD"},"hgnc":{"alias_symbol":["AD023","MGC45027","SLIP1"],"prev_symbol":["MIFD"]},"alphafold":{"accession":"A9UHW6","domains":[{"cath_id":"1.25.40.180","chopping":"9-117","consensus_level":"medium","plddt":93.7208,"start":9,"end":117},{"cath_id":"1.25.40.180","chopping":"123-222","consensus_level":"medium","plddt":93.6901,"start":123,"end":222}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/A9UHW6","model_url":"https://alphafold.ebi.ac.uk/files/AF-A9UHW6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-A9UHW6-F1-predicted_aligned_error_v6.png","plddt_mean":91.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MIF4GD","jax_strain_url":"https://www.jax.org/strain/search?query=MIF4GD"},"sequence":{"accession":"A9UHW6","fasta_url":"https://rest.uniprot.org/uniprotkb/A9UHW6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/A9UHW6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/A9UHW6"}},"corpus_meta":[{"pmid":"8449366","id":"PMC_8449366","title":"Role of a gamete-specific sulfoglycolipid immobilizing protein on mouse sperm-egg binding.","date":"1993","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/8449366","citation_count":69,"is_preprint":false},{"pmid":"18025107","id":"PMC_18025107","title":"SLIP1, a factor required for activation of histone mRNA translation by the stem-loop binding protein.","date":"2007","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18025107","citation_count":63,"is_preprint":false},{"pmid":"20309963","id":"PMC_20309963","title":"Novel interactors and a role for supervillin in early cytokinesis.","date":"2010","source":"Cytoskeleton (Hoboken, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/20309963","citation_count":58,"is_preprint":false},{"pmid":"24336329","id":"PMC_24336329","title":"MIF4G domain containing protein regulates cell cycle and hepatic carcinogenesis by antagonizing CDK2-dependent p27 stability.","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/24336329","citation_count":53,"is_preprint":false},{"pmid":"23804756","id":"PMC_23804756","title":"Structural and biochemical studies of SLIP1-SLBP identify DBP5 and eIF3g as SLIP1-binding proteins.","date":"2013","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/23804756","citation_count":32,"is_preprint":false},{"pmid":"1515145","id":"PMC_1515145","title":"Role of a germ cell-specific sulfolipid-immobilizing protein (SLIP1) in mouse in vivo fertilization.","date":"1992","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/1515145","citation_count":30,"is_preprint":false},{"pmid":"9337084","id":"PMC_9337084","title":"Expression and sulfogalactolipid binding specificity of the recombinant testis-specific cognate heat shock protein 70.","date":"1997","source":"Glycoconjugate journal","url":"https://pubmed.ncbi.nlm.nih.gov/9337084","citation_count":27,"is_preprint":false},{"pmid":"24692427","id":"PMC_24692427","title":"Degradation of oligouridylated histone mRNAs: see UUUUU and goodbye.","date":"2014","source":"Wiley interdisciplinary reviews. RNA","url":"https://pubmed.ncbi.nlm.nih.gov/24692427","citation_count":20,"is_preprint":false},{"pmid":"17014084","id":"PMC_17014084","title":"Folding mechanism of a multiple independently-folding domain protein: double B domain of protein A.","date":"2006","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17014084","citation_count":19,"is_preprint":false},{"pmid":"9444663","id":"PMC_9444663","title":"Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida.","date":"1998","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/9444663","citation_count":19,"is_preprint":false},{"pmid":"22532700","id":"PMC_22532700","title":"INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation.","date":"2012","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/22532700","citation_count":17,"is_preprint":false},{"pmid":"11420386","id":"PMC_11420386","title":"Anti-SLIP1-reactive proteins exist on human spermatozoa and are involved in zona pellucida binding.","date":"2001","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/11420386","citation_count":16,"is_preprint":false},{"pmid":"10456853","id":"PMC_10456853","title":"Role of egg sulfolipidimmobilizing protein 1 on mouse sperm-egg plasma membrane binding.","date":"1999","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/10456853","citation_count":16,"is_preprint":false},{"pmid":"23286197","id":"PMC_23286197","title":"Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1.","date":"2013","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23286197","citation_count":12,"is_preprint":false},{"pmid":"9364447","id":"PMC_9364447","title":"Localization and role of sulfoglycolipid immobilizing protein 1 on the mouse sperm head.","date":"1997","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/9364447","citation_count":8,"is_preprint":false},{"pmid":"39284473","id":"PMC_39284473","title":"Functional characterization of MiFTs implicated in early flowering and stress resistances of mango.","date":"2024","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/39284473","citation_count":5,"is_preprint":false},{"pmid":"39597157","id":"PMC_39597157","title":"Towards a 3D-Printed Millifluidic Device for Investigating Cellular Processes.","date":"2024","source":"Micromachines","url":"https://pubmed.ncbi.nlm.nih.gov/39597157","citation_count":3,"is_preprint":false},{"pmid":"21157122","id":"PMC_21157122","title":"Expression analysis of MIF4GD in the rat testis.","date":"2010","source":"The Journal of reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/21157122","citation_count":2,"is_preprint":false},{"pmid":"14727363","id":"PMC_14727363","title":"[Role of sulfogalactosylglycerolipid and sulfolipidimmobilizing protein 1 in sperm-egg plasma membrane interaction].","date":"2003","source":"Zhonghua nan ke xue = National journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/14727363","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11602,"output_tokens":2772,"usd":0.038193,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9493,"output_tokens":2513,"usd":0.055145,"stage2_stop_reason":"end_turn"},"total_usd":0.093338,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"I need to carefully distinguish between two completely different proteins both called \"SLIP1\":\n\n1. **MIF4GD/SLIP1** (the query gene) = SLBP-Interacting Protein 1, a MIF4G domain-containing translational regulator. This is the canonical human/mammalian gene MIF4GD.\n\n2. **SLIP1** in sperm fertilization papers = Sulfolipid-Immobilizing Protein 1, a sulfoglycolipid-binding sperm protein. This is a completely different protein (appears to be HSP70.2/HSPA2) from the fertilization literature (papers 1, 6, 7, 10, 12, 13, 15, 19).\n\nPapers 1, 6, 7, 10, 12, 13, 15, 19 describe the sperm SLIP1 (sulfoglycolipid immobilizing protein), which is a different gene — this is a symbol collision. I will exclude these.\n\nPaper 9 describes \"MIFD\" but in the context of protein A domain folding — this is a different usage of MIFD (Multiple Independently-Folding Domain). Symbol collision. Exclude.\n\nPaper 16 describes MiFT/MiFD in mango plants — symbol collision. Exclude.\n\nPaper 17 describes millifluidic devices (MiFDs) — not a gene. Exclude.\n\nPapers to include: 2, 3, 4, 5, 8, 11, 14, 18.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"SLIP1 (MIF4GD) was identified as a binding partner of SLBP via yeast two-hybrid screening. Five conserved residues in a 15-amino-acid region of SLBP's N-terminal portion are required for both translation activation and SLIP1 binding; mutations in any of these residues reduce SLIP1 binding. Co-expression of SLIP1 with human SLBP in Xenopus oocytes stimulates translation of a stem-loop reporter mRNA but not a polyadenylated reporter, and siRNA-mediated knockdown of SLIP1 in HeLa cells reduces histone mRNA translation rate and cell viability.\",\n      \"method\": \"Yeast two-hybrid screen, site-directed mutagenesis, Xenopus oocyte translation assay, RNAi knockdown in HeLa cells, GFP reporter assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (two-hybrid, mutagenesis, oocyte functional assay, RNAi with defined translational phenotype) in a single study with clear functional readouts\",\n      \"pmids\": [\"18025107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of zebrafish SLIP1 bound to the translation-activation domain of SLBP was solved at 2.5 Å resolution, defining the molecular determinants of SLBP recognition. A SLIP1-binding motif (SBM) was identified in two additional proteins, eIF3g and the mRNA-export factor DBP5; the 3.25 Å crystal structure of SLIP1 bound to the DBP5 SBM was also determined. Pull-down assays confirmed SLIP1 binding to DBP5 and eIF3g. SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CTIF, suggesting SLIP1 homodimer or SLIP1-CTIF heterodimer can bridge SLBP with SBM-containing proteins in mRNA metabolism.\",\n      \"method\": \"X-ray crystallography (2.5 Å and 3.25 Å resolution structures), pull-down assays, sequence conservation analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — two independent crystal structures plus biochemical pull-down confirmation in a single rigorous study\",\n      \"pmids\": [\"23804756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SLIP1 is a homodimer that does not bind RNA on its own. Unphosphorylated SLBP-SLIP1 forms a 2:2 high-affinity (Kd <0.9 nM) heterotetramer incapable of binding histone mRNA. Phosphorylated SLBP (phosphorylated at 23 Ser/Thr sites) has weak affinity (~3 µM) for SLIP1. Sequential binding — phosphorylated SLBP to the histone mRNA stem-loop, then SLIP1 — is required to form an active ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to a heterodimer. Alanine scanning mutagenesis mapped the SLBP-binding site on SLIP1 to near the dimer interface; a single-point mutant there abolished SLBP interaction in vitro and reduced histone mRNA abundance in vivo.\",\n      \"method\": \"Biophysical characterization (sedimentation, binding assays), baculovirus phosphorylated SLBP expression, alanine scanning mutagenesis, in vivo histone mRNA measurement\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with phosphorylated and unphosphorylated proteins, quantitative binding measurements, mutagenesis with in vivo validation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"23286197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"INT6/EIF3E (an eIF3 subunit) was identified as a binding partner of MIF4GD/SLIP1 via yeast two-hybrid screen. INT6 and MIF4GD co-localize in cytoplasmic foci. siRNA knockdown of INT6 reduces S-phase histone mRNA translation, assessed by endogenous histone protein levels and luciferase reporter driven by histone stem-loop elements; INT6 overexpression has the opposite effect. INT6 interacts with both MIF4GD and SLBP, placing it in the histone mRNA translation complex.\",\n      \"method\": \"Yeast two-hybrid screen, RNAi knockdown, luciferase reporter assay, co-localization by immunofluorescence\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid plus RNAi phenotype plus co-localization, single lab, multiple methods but no reciprocal Co-IP reported in abstract\",\n      \"pmids\": [\"22532700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MIF4GD was identified as a novel binding partner for the CDK inhibitor p27(kip1) via yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation and GST pull-down assays. MIF4GD interaction with p27 stabilizes p27 in both the nucleus and cytoplasm by suppressing CDK2-mediated phosphorylation of p27 at Thr187. MIF4GD overexpression increases p27 levels and reduces cell proliferation; MIF4GD knockdown promotes cell cycle progression with decreased p27 levels. MIF4GD overexpression reduces colony formation and inhibits xenograft tumor growth in nude mice.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, GST pull-down, overexpression and knockdown in hepatocellular carcinoma cells, xenograft tumor model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and GST pull-down confirming interaction, plus loss- and gain-of-function with defined cell cycle phenotype, single lab\",\n      \"pmids\": [\"24336329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MIF4GD/SLIP1 was identified as a candidate binding partner of supervillin (via yeast two-hybrid screen); the MIF4GD-interacting sequence co-localizes with and mis-localizes EGFP-supervillin in mammalian cells, and MIF4GD-interacting sequences mimic supervillin overexpression by inhibiting cell spreading.\",\n      \"method\": \"Yeast two-hybrid screen, co-localization of EGFP-supervillin with interacting sequences in mammalian cells, cell spreading assay\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid identification with co-localization in cells, single lab, no direct biochemical confirmation of MIF4GD-supervillin interaction reported for this pair\",\n      \"pmids\": [\"20309963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In rat testis, MIF4GD exists in three isoforms (25, 20, and 16 kDa). The 20-kDa form is testis-specific. The 16-kDa MIF4GD uniquely co-fractionates with 40S ribosomal subunits and ribosomes in spermatogenic cells by subcellular fractionation, suggesting it functions as a translational regulator in spermiogenesis.\",\n      \"method\": \"Western blot, subcellular fractionation (ribosome sedimentation), tissue expression analysis\",\n      \"journal\": \"The Journal of reproduction and development\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — fractionation-based localization without functional consequence directly tested, single lab, single method\",\n      \"pmids\": [\"21157122\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MIF4GD (SLIP1) is a MIF4G domain-containing homodimer that functions as a translational activator of replication-dependent histone mRNAs by binding the translation-activation domain of SLBP (stem-loop binding protein); the active complex requires sequential binding of phosphorylated SLBP to the histone mRNA stem-loop followed by SLIP1 recruitment, forming a ternary complex that bridges the 3' stem-loop to the translation initiation machinery via interactions with eIF3g and mRNA-export factor DBP5, and is further regulated by INT6/EIF3E; MIF4GD also interacts with p27(kip1) to suppress CDK2-mediated phosphorylation of p27 at Thr187, thereby stabilizing p27 and restraining cell cycle progression.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MIF4GD (SLIP1) is a MIF4G-domain homodimer that functions as a translational activator of replication-dependent histone mRNAs by coupling the stem-loop binding protein SLBP to the translation initiation machinery [#0, #2]. It was identified as an SLBP partner whose binding requires a conserved 15-residue translation-activation region in the SLBP N-terminus, and its depletion lowers histone mRNA translation and cell viability [#0]. Activation is assembled sequentially and is phosphorylation-gated: unphosphorylated SLBP and SLIP1 form a tight 2:2 heterotetramer that cannot bind RNA, whereas phosphorylated SLBP first binds the histone mRNA stem-loop and then recruits SLIP1, forming the active ternary complex, with SLBP Thr171 phosphorylation driving heterotetramer-to-heterodimer conversion [#2]. Crystal structures of SLIP1 bound to SLBP and to a SLIP1-binding motif (SBM) define how the homodimer simultaneously engages SLBP and SBM-containing factors eIF3g and the mRNA-export factor DBP5, bridging the histone mRNA to initiation; the homodimerization and SBM-binding residues are shared with the MIF4G domain of CTIF [#1]. The eIF3 subunit INT6/EIF3E binds both MIF4GD and SLBP and co-localizes with MIF4GD in cytoplasmic foci, promoting S-phase histone mRNA translation [#3]. Independently of its translational role, MIF4GD binds the CDK inhibitor p27(kip1), suppresses CDK2-mediated phosphorylation of p27 at Thr187 to stabilize p27 in nucleus and cytoplasm, and thereby restrains proliferation, reducing colony formation and xenograft tumor growth [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established MIF4GD/SLIP1 as a dedicated activator of histone mRNA translation by identifying it as the SLBP partner that links the SLBP translation-activation domain to a functional output.\",\n      \"evidence\": \"Yeast two-hybrid screen, SLBP mutagenesis, Xenopus oocyte stem-loop reporter translation assay, and RNAi in HeLa cells\",\n      \"pmids\": [\"18025107\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not define the structural basis of the SLBP-SLIP1 interface\",\n        \"Connection to the core initiation machinery not yet identified\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"First indications of additional partners and tissue-specific forms, hinting at roles beyond the canonical SLBP complex.\",\n      \"evidence\": \"Yeast two-hybrid with supervillin plus cell-spreading assay; Western blot and ribosome fractionation of rat testis isoforms\",\n      \"pmids\": [\"20309963\", \"21157122\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Supervillin interaction not confirmed biochemically and functional relevance unestablished\",\n        \"Testis-specific 20-kDa isoform function not tested\",\n        \"Ribosome co-fractionation of the 16-kDa form is correlative, no functional consequence shown\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed an eIF3 subunit, INT6/EIF3E, within the histone mRNA translation complex, providing a route from SLIP1/SLBP to the initiation apparatus.\",\n      \"evidence\": \"Yeast two-hybrid, RNAi and overexpression with histone stem-loop luciferase reporter, immunofluorescence co-localization\",\n      \"pmids\": [\"22532700\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No reciprocal Co-IP reported confirming the MIF4GD-INT6 interaction\",\n        \"Mechanism by which INT6 enhances translation not resolved\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the assembly logic and structural determinants of the activation complex, showing SLIP1 is a non-RNA-binding homodimer whose recruitment is gated by SLBP phosphorylation and that it bridges SBM-containing factors eIF3g and DBP5.\",\n      \"evidence\": \"Crystal structures of SLIP1-SLBP and SLIP1-DBP5 SBM, pull-downs, biophysical reconstitution with phosphorylated/unphosphorylated SLBP, alanine scanning with in vivo histone mRNA readout\",\n      \"pmids\": [\"23804756\", \"23286197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In-cell stoichiometry and dynamics of the heterotetramer-to-ternary transition not directly observed\",\n        \"Whether SLIP1-CTIF heterodimers form in vivo not established\",\n        \"Role of DBP5 in histone mRNA translation not functionally tested\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed a translation-independent role in cell cycle control through stabilization of the CDK inhibitor p27, linking MIF4GD to proliferation restraint and tumor suppression.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP and GST pull-down, gain/loss-of-function in hepatocellular carcinoma cells, xenograft tumor model\",\n      \"pmids\": [\"24336329\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which MIF4GD suppresses CDK2-mediated p27 Thr187 phosphorylation not defined\",\n        \"Relationship between the translational and p27-stabilizing functions unresolved\",\n        \"Single lab, single tumor context\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MIF4GD partitions between its histone mRNA translation role and its p27-stabilizing cell cycle role, and whether these activities are coordinated, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No unifying model connecting translational activation and p27 stabilization\",\n        \"Regulation of MIF4GD itself (modifications, localization control) uncharacterized\",\n        \"In vivo physiological requirement not addressed by knockout\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-72766\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SLBP\", \"EIF3E\", \"EIF3G\", \"DBP5\", \"CDKN1B\", \"CTIF\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}