{"gene":"RASSF1","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2000,"finding":"RASSF1 binds Ras in a GTP-dependent manner both in vivo and directly in vitro, and functions as a Ras effector that mediates apoptosis; activated Ras enhances RASSF1-induced cell death while dominant negative Ras inhibits it, and the cell death is caspase-dependent.","method":"In vitro pulldown, co-immunoprecipitation, transient transfection with dominant-active/dominant-negative Ras, caspase inhibitor rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — direct in vitro binding plus in vivo genetic epistasis with multiple orthogonal methods","pmids":["10998413"],"is_preprint":false},{"year":2004,"finding":"Endogenous MST1 co-immunoprecipitates with RASSF1A and NORE1A from KB cells; co-expression of RASSF1A suppresses MST1 Thr183 autophosphorylation and abolishes Mg-ATP-mediated MST1 autoactivation in vitro, acting as an inhibitor of MST1/2 kinase activation. However, membrane-targeted NORE1A or Ras(G12V)-recruited MST1 shows increased Thr183 phosphorylation, indicating context-dependent activation.","method":"Co-immunoprecipitation of endogenous proteins, in vitro kinase autoactivation assay, in vitro addition of purified recombinant proteins, co-transfection with CAAX-tagged or myristoylated constructs","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro kinase assay combined with endogenous co-IP and mutagenesis","pmids":["15109305"],"is_preprint":false},{"year":2004,"finding":"RASSF1 (region 74-123 of RASSF1C / 144-193 of RASSF1A) physically interacts with the second intracellular loop (residues 652-748) of plasma membrane Ca2+-ATPase PMCA4b; this interaction was identified by yeast two-hybrid, confirmed by co-immunoprecipitation, immunofluorescence co-localization, and GST pulldown. Functional consequence: co-expression of PMCA4b and RASSF1 inhibits EGF-dependent ERK pathway activation, and this inhibition is abolished by a competing peptide that blocks PMCA/RASSF1 association.","method":"Yeast two-hybrid screen, co-immunoprecipitation, GST pulldown, immunofluorescence co-localization, ERK pathway activity assay with competing peptide","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal binding assays plus functional rescue with competing peptide","pmids":["15145946"],"is_preprint":false},{"year":2005,"finding":"Both RASSF1A and RASSF1C localize to mitochondria and microtubules and exhibit paclitaxel-like microtubule hyperstabilization and disruption of mitosis. When co-expressed with C19ORF5 (a MAP1B homologue that accumulates on hyperstabilized microtubules and causes cell death), the unique N-terminal sequence of RASSF1C prevents microtubule hyperstabilization, conferring specificity on RASSF1A for this function.","method":"Subcellular localization by immunofluorescence/GFP tagging, co-expression and microtubule stabilization assays, interaction with C19ORF5","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct localization and functional co-expression experiments in a single study","pmids":["15753381"],"is_preprint":false},{"year":2002,"finding":"C19ORF5 interacts with RASSF1 (identified by yeast two-hybrid interaction cloning); GFP-tagged RASSF1 co-localizes with mitochondria and beta-tubulin in the cytosol during interphase and reorganizes around separating chromosomes during mitosis.","method":"Yeast two-hybrid interaction cloning, GFP-tagging and immunofluorescence co-localization","journal":"In vitro cellular & developmental biology. Animal","confidence":"Medium","confidence_rationale":"Tier 3 — yeast two-hybrid plus co-localization, single study","pmids":["12762840"],"is_preprint":false},{"year":2005,"finding":"RASSF1A null mouse embryonic fibroblasts display increased sensitivity to microtubule-depolymerizing agents, establishing a role for RASSF1A in microtubule stability in vivo. Rassf1A null mice show increased spontaneous and irradiation-induced tumorigenesis.","method":"Targeted gene deletion in mouse, microtubule depolymerization sensitivity assay in primary fibroblasts, tumor incidence monitoring","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — clean genetic knockout with defined molecular and phenotypic readouts","pmids":["16135822"],"is_preprint":false},{"year":2006,"finding":"RASSF1/NORE1 polypeptides bind MST1/2 through their SARAH domains; recombinant MST1/2 form spontaneous dimers and autoactivate in vitro by intradimer transphosphorylation of the activation loop (Thr183/Thr180); NORE1/RASSF polypeptides inhibit this autoactivation. The C. elegans RASSF ortholog (T24F1.3) also binds MST1/2 via SARAH domains.","method":"In vitro kinase reconstitution, SARAH domain binding assays, cross-species comparison","journal":"Methods in enzymology","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro kinase assay with domain mapping; confirms prior Biochemical Journal findings","pmids":["16757333"],"is_preprint":false},{"year":2011,"finding":"Rassf1 interacts with Daxx (co-immunoprecipitation) and partially co-localizes with Daxx during mitosis. Rassf1/Daxx depletion or expression of the Daxx-binding domain of Rassf1 elevates cyclin B stability and increases taxol resistance, defining a mitotic stress checkpoint. Rassf1 depletion does not alter APC/C ubiquitin ligase activity in vitro, indicating the checkpoint requires the mitotic cellular environment.","method":"Co-immunoprecipitation, RNAi knockdown, cyclin B stability assay, in vitro APC/C ubiquitin ligase assay, time-lapse microscopy, mouse xenograft","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP plus in vitro APC/C assay and in vivo xenograft, single lab","pmids":["21643015"],"is_preprint":false},{"year":2013,"finding":"RASSF family members (RASSF1-6) interact with MST kinases through SARAH domain-mediated interactions, confirmed by in vitro interaction studies; RASSF7 was identified as a new MST kinase interacting partner. RA domain interactions show diversity across RASSF members. RASSF proteins act as adaptors assembling multiple protein complexes.","method":"In vitro interaction studies (SARAH and RA domain), structural modeling, in silico prediction","journal":"Advances in biological regulation","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vitro binding assays with domain mapping; single study","pmids":["23357313"],"is_preprint":false},{"year":2015,"finding":"RASSF1A restricts SRC kinase activity and prevents motility/invasion, while the RASSF1C isoform (expressed when RASSF1A promoter is methylated) targets SRC/YES kinases to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A promoter methylation reduces YAP phospho-S127, derepressing YAP1; this is identified by affinity proteomics, proximity ligation assay, and real-time molecular visualization.","method":"Affinity proteomics, proximity ligation assay, live-cell imaging, phosphorylation assays, in vitro and in vivo invasion/motility assays, promoter methylation analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (proteomics, PLA, imaging, functional assays) in a single study","pmids":["26549256"],"is_preprint":false},{"year":2017,"finding":"miR-193a-3p directly binds to the RASSF1 3'UTR and represses RASSF1 mRNA and protein expression; excess miR-193a-3p impairs the Rassf1-Syntaxin 16 signalling pathway required for cytokinesis completion, causing polyploidy, multipolar mitotic spindles, and elevated cell death.","method":"Dual luciferase reporter assay, immunoblotting, qRT-PCR, time-lapse microscopy, immunofluorescence","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — luciferase validation of direct miRNA-3'UTR interaction plus functional cell division phenotype","pmids":["28449010"],"is_preprint":false},{"year":2019,"finding":"RASSF1-AS1 (an antisense lncRNA from the RASSF1A locus) partially overlaps RASSF1A mRNA at exon 2 and directly binds RASSF1A mRNA, suppressing its translation at the post-transcriptional level. RNA pulldown and luciferase assays confirm this direct binding. Mutated RASSF1-AS1 lacking the binding region has no effect on RASSF1A expression or NF-κB activation.","method":"RNA pulldown, luciferase activity assay, overexpression/knockdown in mouse primary cardiac fibroblasts, Western blot","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 2 — RNA pulldown plus reporter assay with domain deletion mutant, single lab","pmids":["30571844"],"is_preprint":false},{"year":2016,"finding":"miR-181a directly targets RASSF1 (validated by luciferase assay), reducing RASSF1 expression; knockdown of RASSF1 increases sorafenib resistance in hepatocellular carcinoma cells, linking RASSF1 to MAPK signaling and sorafenib sensitivity.","method":"Luciferase reporter assay, miRNA overexpression/inhibition, RNAi knockdown, apoptosis assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2-3 — luciferase validation plus functional knockdown, single lab","pmids":["27384977"],"is_preprint":false},{"year":2016,"finding":"rhBMP-2 increases MST1, MOB1, and p-YAP levels in esophageal cancer cells, and RASSF1 binding to MST1 is enhanced upon rhBMP-2 treatment. RASSF1 knockdown reverses the p-YAP induction by rhBMP-2, placing RASSF1 upstream of MST1 in rhBMP-2-activated Hippo signaling.","method":"Co-immunoprecipitation (RASSF1-MST1), siRNA knockdown, Western blot, MTT assay, xenograft","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP combined with knockdown rescue in a single study","pmids":["27230238"],"is_preprint":false},{"year":2023,"finding":"ATF4 directly binds to ATF4-binding sites in the RASSF1 promoter (shown by ChIP) and activates RASSF1 transcription (shown by reporter assay). RASSF1 ablation mitigates expression of the ATF4 effector BBC3 and abrogates tunicamycin-induced apoptosis, placing RASSF1 downstream of ATF4 in ER stress-associated apoptosis.","method":"Chromatin immunoprecipitation (ChIP), luciferase reporter assay, RASSF1 knockdown, tunicamycin-induced apoptosis assay","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus reporter assay plus functional KO phenotype, single lab","pmids":["36723232"],"is_preprint":false},{"year":2024,"finding":"P. multocida infection upregulates Rassf1 expression in pulmonary epithelial cells; Rassf1 enhances Hippo-YAP pathway through phosphorylation (increased p-MST1/2, p-LATS1, p-YAP). In vitro knockdown of Rassf1 enhances YAP activity and reduces apoptosis during infection, and pharmacological Hippo inhibition rescues apoptosis in vitro and reduces lung injury in vivo.","method":"RNA-seq, siRNA knockdown, Western blot for pathway phosphorylation, mouse and rabbit infection models, pharmacological inhibition (XMU-MP-1)","journal":"Veterinary research","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro KD with defined phosphorylation readout plus in vivo model; single lab","pmids":["38493147"],"is_preprint":false},{"year":2025,"finding":"RFX2 transcription factor binds directly to the RASSF1 promoter (shown by dual-luciferase and ChIP assays) and activates RASSF1 transcription. RASSF1 knockdown reverses the ability of RFX2 overexpression to inhibit immune escape, placing RASSF1 downstream of RFX2 in Hippo/YAP-mediated immune regulation.","method":"Dual-luciferase reporter assay, ChIP-PCR, siRNA knockdown, co-culture with CD8+ T cells, Western blot for YAP phosphorylation","journal":"Cell division","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus reporter assay with functional rescue, single lab","pmids":["40069841"],"is_preprint":false}],"current_model":"RASSF1A functions as a scaffold/adaptor tumor suppressor that binds Ras-GTP through its RA domain and MST1/2 kinases through its SARAH domain; it inhibits MST1/2 autoactivation in quiescent cells while directing them to sites of activation, stabilizes microtubules, restricts SRC kinase activity at cell-cell junctions, and promotes apoptosis downstream of oncogenic Ras and ER stress (ATF4), whereas the alternative RASSF1C isoform has opposing pro-tumorigenic effects including SRC/YES activation and YAP1 derepression upon epigenetic silencing of RASSF1A."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing RASSF1 as a direct Ras effector answered the question of whether RAS-family GTPases signal through RASSF proteins and showed that RASSF1-mediated apoptosis is Ras-dependent and caspase-dependent.","evidence":"In vitro pulldown, co-IP, and epistasis with dominant-active/dominant-negative Ras in transfected cells","pmids":["10998413"],"confidence":"High","gaps":["Structural basis of GTP-dependent Ras–RASSF1 interaction not determined","Endogenous Ras–RASSF1 complex not demonstrated","Downstream caspase cascade not mapped"]},{"year":2002,"claim":"Identification of C19ORF5 as a RASSF1 interactor and visualization of RASSF1 on microtubules and mitochondria revealed that RASSF1 operates at the cytoskeleton and mitochondria, not solely in cytosolic Ras signaling.","evidence":"Yeast two-hybrid screen and GFP co-localization with β-tubulin and mitochondria","pmids":["12762840"],"confidence":"Medium","gaps":["Functional significance of mitochondrial localization unclear","Direct binding to tubulin not demonstrated"]},{"year":2004,"claim":"Demonstration that RASSF1A inhibits MST1 autoactivation in vitro yet permits activation when recruited to membranes established the dual-function scaffold model: RASSF1A restrains MST1/2 in the cytoplasm but enables context-dependent activation at membranes.","evidence":"Endogenous co-IP from KB cells, reconstituted in vitro kinase assays with purified proteins, CAAX-targeted constructs","pmids":["15109305"],"confidence":"High","gaps":["In vivo physiological membrane-targeting signal unknown","Stoichiometry of RASSF1A–MST1 complex not quantified"]},{"year":2004,"claim":"Discovery of the RASSF1–PMCA4b interaction at the plasma membrane, which inhibits EGF-driven ERK signaling, revealed a non-Ras mechanism by which RASSF1 limits MAPK activation.","evidence":"Yeast two-hybrid, co-IP, GST pulldown, and ERK activity rescue with competing peptide","pmids":["15145946"],"confidence":"High","gaps":["Whether PMCA4b–RASSF1 interaction is isoform-specific (RASSF1A vs 1C) not resolved","In vivo relevance in animal models not tested"]},{"year":2005,"claim":"Rassf1A knockout mice showed increased tumorigenesis and MEF hypersensitivity to microtubule-destabilizing agents, providing genetic proof that RASSF1A functions as a tumor suppressor through microtubule stabilization in vivo.","evidence":"Targeted gene deletion in mouse, primary fibroblast drug sensitivity assays, tumor monitoring","pmids":["16135822","15753381"],"confidence":"High","gaps":["Whether microtubule stabilization or Hippo regulation is the primary tumor-suppressive mechanism not distinguished","Tissue-specific contributions not dissected"]},{"year":2011,"claim":"Identification of the RASSF1–Daxx interaction and its role in a mitotic checkpoint that regulates cyclin B stability linked RASSF1 to mitotic fidelity beyond simple microtubule stabilization.","evidence":"Co-IP, RNAi, time-lapse microscopy, cyclin B stability assays, in vitro APC/C assay","pmids":["21643015"],"confidence":"Medium","gaps":["Mechanism by which RASSF1–Daxx promotes cyclin B degradation without altering APC/C activity in vitro not explained","Single-lab finding; independent replication absent"]},{"year":2015,"claim":"Affinity proteomics and proximity ligation revealed that RASSF1A restricts SRC activity while RASSF1C activates SRC/YES at cell junctions, explaining how promoter methylation-driven isoform switching derepresses YAP1 and promotes invasion.","evidence":"Affinity proteomics, PLA, live-cell imaging, phosphorylation assays in epithelial cells","pmids":["26549256"],"confidence":"High","gaps":["Direct binding interface between RASSF1C and SRC/YES not mapped","Whether RASSF1C activates YAP independently of MST1/2 not resolved"]},{"year":2016,"claim":"Placing RASSF1 upstream of MST1–YAP in BMP-2 signaling in esophageal cancer cells extended the RASSF1–Hippo axis to growth factor contexts beyond canonical Ras.","evidence":"Co-IP of RASSF1–MST1 under BMP-2 treatment, siRNA knockdown, xenograft","pmids":["27230238"],"confidence":"Medium","gaps":["Single co-IP without reciprocal validation under BMP-2","Whether BMP-2 triggers RASSF1A membrane recruitment not tested"]},{"year":2023,"claim":"ChIP and reporter assays showing ATF4 directly activates RASSF1 transcription, and that RASSF1 loss abrogates tunicamycin-induced apoptosis, established RASSF1 as a transcriptional target of the integrated stress response mediating ER stress-induced cell death.","evidence":"ChIP, luciferase reporter, RASSF1 knockdown, tunicamycin-induced apoptosis","pmids":["36723232"],"confidence":"Medium","gaps":["Whether RASSF1A or RASSF1C is the isoform induced by ATF4 not specified","Downstream effector pathway (Hippo vs other) linking RASSF1 to BBC3 not resolved"]},{"year":2024,"claim":"Demonstration that Rassf1 activates the full Hippo phosphorylation cascade (p-MST1/2 → p-LATS1 → p-YAP) during bacterial infection-induced apoptosis in pulmonary epithelial cells extended RASSF1–Hippo signaling to innate immune and infection contexts.","evidence":"RNA-seq, siRNA knockdown, phospho-Western blots, mouse/rabbit infection models, XMU-MP-1 rescue","pmids":["38493147"],"confidence":"Medium","gaps":["Direct physical interaction between RASSF1 and MST1/2 not confirmed in pulmonary epithelial cells","Whether RASSF1 acts cell-autonomously or through paracrine signaling not distinguished"]},{"year":null,"claim":"Key unresolved questions include: the structural basis for RASSF1A's dual role as MST1/2 inhibitor in the cytoplasm versus activator at membranes; the relative contributions of microtubule stabilization, Hippo activation, and SRC restriction to tumor suppression; and the mechanism by which RASSF1C opposes RASSF1A function at the molecular level.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of RASSF1A–MST1 SARAH domain complex","Genetic separation of microtubule-stabilizing vs Hippo-activating functions not achieved in vivo","Biochemical basis by which RASSF1C activates SRC/YES while RASSF1A inhibits it not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,6,9,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,6,9,15]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3,4,5]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,9]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,14,15]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,6,9,13,15]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7,10]}],"complexes":[],"partners":["MST1","MST2","DAXX","ATP2B4","SRC","YES1","C19ORF5"],"other_free_text":[]},"mechanistic_narrative":"RASSF1 is a Ras effector and scaffold protein that integrates Ras-GTP signaling, Hippo pathway regulation, and microtubule dynamics to exert tumor-suppressive functions. It binds Ras in a GTP-dependent manner through its RA domain and mediates caspase-dependent apoptosis downstream of activated Ras, ATF4-driven ER stress, and BMP-2 signaling [PMID:10998413, PMID:36723232, PMID:27230238]. Through its SARAH domain, RASSF1A interacts with MST1/2 kinases, restraining their autoactivation in quiescent cells while facilitating context-dependent Hippo pathway activation that promotes YAP phosphorylation and suppresses proliferation [PMID:15109305, PMID:16757333, PMID:38493147]. RASSF1A additionally stabilizes microtubules, cooperates with Daxx in a mitotic checkpoint controlling cyclin B turnover, and restricts SRC kinase activity at cell–cell junctions; loss of RASSF1A by promoter methylation shifts signaling toward the RASSF1C isoform, which activates SRC/YES and derepresses YAP1 to promote invasion [PMID:16135822, PMID:21643015, PMID:26549256]."},"prefetch_data":{"uniprot":{"accession":"Q9NS23","full_name":"Ras association domain-containing protein 1","aliases":[],"length_aa":344,"mass_kda":39.2,"function":"Potential tumor suppressor. Required for death receptor-dependent apoptosis. Mediates activation of STK3/MST2 and STK4/MST1 during Fas-induced apoptosis by preventing their dephosphorylation. When associated with MOAP1, promotes BAX conformational change and translocation to mitochondrial membranes in response to TNF and TNFSF10 stimulation. Isoform A interacts with CDC20, an activator of the anaphase-promoting complex, APC, resulting in the inhibition of APC activity and mitotic progression. Inhibits proliferation by negatively regulating cell cycle progression at the level of G1/S-phase transition by regulating accumulation of cyclin D1 protein. Isoform C has been shown not to perform these roles, no function has been identified for this isoform. Isoform A disrupts interactions among MDM2, DAXX and USP7, thus contributing to the efficient activation of TP53 by promoting MDM2 self-ubiquitination in cell-cycle checkpoint control in response to DNA damage","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NS23/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RASSF1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"STK4","stoichiometry":0.2},{"gene":"VAPA","stoichiometry":0.2},{"gene":"VAPB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RASSF1","total_profiled":1310},"omim":[{"mim_id":"612620","title":"RAS ASSOCIATION DOMAIN FAMILY, MEMBER 6; RASSF6","url":"https://www.omim.org/entry/612620"},{"mim_id":"610559","title":"RAS ASSOCIATION DOMAIN FAMILY, MEMBER 4; RASSF4","url":"https://www.omim.org/entry/610559"},{"mim_id":"609492","title":"RAS ASSOCIATION DOMAIN FAMILY PROTEIN 2; RASSF2","url":"https://www.omim.org/entry/609492"},{"mim_id":"607020","title":"RAS ASSOCIATION DOMAIN FAMILY PROTEIN 5; RASSF5","url":"https://www.omim.org/entry/607020"},{"mim_id":"607019","title":"RAS ASSOCIATION DOMAIN FAMILY PROTEIN 3; RASSF3","url":"https://www.omim.org/entry/607019"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RASSF1"},"hgnc":{"alias_symbol":["NORE2A","REH3P21","RDA32","123F2"],"prev_symbol":[]},"alphafold":{"accession":"Q9NS23","domains":[{"cath_id":"3.10.20.90","chopping":"49-121_134-182_205-290","consensus_level":"medium","plddt":79.6163,"start":49,"end":290},{"cath_id":"1.20.5","chopping":"301-341","consensus_level":"high","plddt":85.4337,"start":301,"end":341}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NS23","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NS23-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NS23-F1-predicted_aligned_error_v6.png","plddt_mean":71.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RASSF1","jax_strain_url":"https://www.jax.org/strain/search?query=RASSF1"},"sequence":{"accession":"Q9NS23","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NS23.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NS23/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NS23"}},"corpus_meta":[{"pmid":"15109305","id":"PMC_15109305","title":"Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras.","date":"2004","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15109305","citation_count":303,"is_preprint":false},{"pmid":"10998413","id":"PMC_10998413","title":"Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10998413","citation_count":247,"is_preprint":false},{"pmid":"16135822","id":"PMC_16135822","title":"The RASSF1A isoform of RASSF1 promotes microtubule stability and suppresses tumorigenesis.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16135822","citation_count":92,"is_preprint":false},{"pmid":"15145946","id":"PMC_15145946","title":"Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1).","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15145946","citation_count":89,"is_preprint":false},{"pmid":"11965544","id":"PMC_11965544","title":"RASSF3 and NORE1: identification and cloning of two human homologues of the putative tumor suppressor gene RASSF1.","date":"2002","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/11965544","citation_count":88,"is_preprint":false},{"pmid":"19795445","id":"PMC_19795445","title":"PTEN, RASSF1 and DAPK site-specific hypermethylation and outcome in surgically treated stage I and II nonsmall cell lung cancer patients.","date":"2010","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/19795445","citation_count":79,"is_preprint":false},{"pmid":"32104074","id":"PMC_32104074","title":"CircHIPK3 Promotes Gemcitabine (GEM) Resistance in Pancreatic Cancer Cells by Sponging miR-330-5p and Targets RASSF1.","date":"2020","source":"Cancer management and 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Rassf1-Hippo-Yap pathway to induce pulmonary epithelial apoptosis.","date":"2024","source":"Veterinary research","url":"https://pubmed.ncbi.nlm.nih.gov/38493147","citation_count":11,"is_preprint":false},{"pmid":"30571844","id":"PMC_30571844","title":"RASSF1-AS1, an antisense lncRNA of RASSF1A, inhibits the translation of RASSF1A to exacerbate cardiac fibrosis in mice.","date":"2019","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/30571844","citation_count":11,"is_preprint":false},{"pmid":"29778234","id":"PMC_29778234","title":"EGFL7 and RASSF1 promoter hypermethylation in epithelial ovarian cancer.","date":"2018","source":"Cancer genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29778234","citation_count":10,"is_preprint":false},{"pmid":"31595551","id":"PMC_31595551","title":"RASSF1 promotes cardiomyocyte apoptosis after acute myocardial infarction and is regulated by miR-125b.","date":"2019","source":"Journal of cellular 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Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban","url":"https://pubmed.ncbi.nlm.nih.gov/19821102","citation_count":5,"is_preprint":false},{"pmid":"36584330","id":"PMC_36584330","title":"Promoter methylation levels of RASSF1 and ATIC genes are associated with lung cancer in Iranian patients.","date":"2023","source":"Hormone molecular biology and clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/36584330","citation_count":4,"is_preprint":false},{"pmid":"36648833","id":"PMC_36648833","title":"An Optimized CoBRA Method for the Microfluidic Electrophoresis Detection of Breast Cancer Associated RASSF1 Methylation.","date":"2023","source":"Biotech (Basel (Switzerland))","url":"https://pubmed.ncbi.nlm.nih.gov/36648833","citation_count":4,"is_preprint":false},{"pmid":"36177104","id":"PMC_36177104","title":"Cell-free methylation of RASSF1 and CDKN2AIP genes in the diagnosis of hepatocellular carcinoma associated with hepatitis B virus cirrhosis.","date":"2022","source":"Hepatology forum","url":"https://pubmed.ncbi.nlm.nih.gov/36177104","citation_count":3,"is_preprint":false},{"pmid":"16329043","id":"PMC_16329043","title":"Molecular alterations of monophasic synovial sarcoma: loss of chromosome 3p does not alter RASSF1 and MLH1 transcriptional activity.","date":"2006","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/16329043","citation_count":3,"is_preprint":false},{"pmid":"31578881","id":"PMC_31578881","title":"Circulating tumor DNA RASSF1 methylation for predicting cancer risk: a diagnostic meta-analysis.","date":"2019","source":"Future oncology (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/31578881","citation_count":2,"is_preprint":false},{"pmid":"28026816","id":"PMC_28026816","title":"[GSTP1, APC and RASSF1 gene methylation in prostate cancer samples: comparative analysis of MS-HRM method and Infinium HumanMethylation450 BeadChip beadchiparray diagnostic value].","date":"2016","source":"Biomeditsinskaia khimiia","url":"https://pubmed.ncbi.nlm.nih.gov/28026816","citation_count":2,"is_preprint":false},{"pmid":"40069841","id":"PMC_40069841","title":"RFX2 downregulates RASSF1 expression and YAP phosphorylation through Hippo signaling to promote immune escape in lung adenocarcinoma.","date":"2025","source":"Cell division","url":"https://pubmed.ncbi.nlm.nih.gov/40069841","citation_count":1,"is_preprint":false},{"pmid":"40645298","id":"PMC_40645298","title":"Ras association domain family member 1 (RASSF1): Molecular characteristics, clinical relevance and therapeutic interventions in cancer.","date":"2025","source":"Biochimica et biophysica acta. 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However, membrane-targeted NORE1A or Ras(G12V)-recruited MST1 shows increased Thr183 phosphorylation, indicating context-dependent activation.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, in vitro kinase autoactivation assay, in vitro addition of purified recombinant proteins, co-transfection with CAAX-tagged or myristoylated constructs\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro kinase assay combined with endogenous co-IP and mutagenesis\",\n      \"pmids\": [\"15109305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RASSF1 (region 74-123 of RASSF1C / 144-193 of RASSF1A) physically interacts with the second intracellular loop (residues 652-748) of plasma membrane Ca2+-ATPase PMCA4b; this interaction was identified by yeast two-hybrid, confirmed by co-immunoprecipitation, immunofluorescence co-localization, and GST pulldown. Functional consequence: co-expression of PMCA4b and RASSF1 inhibits EGF-dependent ERK pathway activation, and this inhibition is abolished by a competing peptide that blocks PMCA/RASSF1 association.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, GST pulldown, immunofluorescence co-localization, ERK pathway activity assay with competing peptide\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal binding assays plus functional rescue with competing peptide\",\n      \"pmids\": [\"15145946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Both RASSF1A and RASSF1C localize to mitochondria and microtubules and exhibit paclitaxel-like microtubule hyperstabilization and disruption of mitosis. When co-expressed with C19ORF5 (a MAP1B homologue that accumulates on hyperstabilized microtubules and causes cell death), the unique N-terminal sequence of RASSF1C prevents microtubule hyperstabilization, conferring specificity on RASSF1A for this function.\",\n      \"method\": \"Subcellular localization by immunofluorescence/GFP tagging, co-expression and microtubule stabilization assays, interaction with C19ORF5\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct localization and functional co-expression experiments in a single study\",\n      \"pmids\": [\"15753381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"C19ORF5 interacts with RASSF1 (identified by yeast two-hybrid interaction cloning); GFP-tagged RASSF1 co-localizes with mitochondria and beta-tubulin in the cytosol during interphase and reorganizes around separating chromosomes during mitosis.\",\n      \"method\": \"Yeast two-hybrid interaction cloning, GFP-tagging and immunofluorescence co-localization\",\n      \"journal\": \"In vitro cellular & developmental biology. Animal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid plus co-localization, single study\",\n      \"pmids\": [\"12762840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RASSF1A null mouse embryonic fibroblasts display increased sensitivity to microtubule-depolymerizing agents, establishing a role for RASSF1A in microtubule stability in vivo. Rassf1A null mice show increased spontaneous and irradiation-induced tumorigenesis.\",\n      \"method\": \"Targeted gene deletion in mouse, microtubule depolymerization sensitivity assay in primary fibroblasts, tumor incidence monitoring\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic knockout with defined molecular and phenotypic readouts\",\n      \"pmids\": [\"16135822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RASSF1/NORE1 polypeptides bind MST1/2 through their SARAH domains; recombinant MST1/2 form spontaneous dimers and autoactivate in vitro by intradimer transphosphorylation of the activation loop (Thr183/Thr180); NORE1/RASSF polypeptides inhibit this autoactivation. The C. elegans RASSF ortholog (T24F1.3) also binds MST1/2 via SARAH domains.\",\n      \"method\": \"In vitro kinase reconstitution, SARAH domain binding assays, cross-species comparison\",\n      \"journal\": \"Methods in enzymology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro kinase assay with domain mapping; confirms prior Biochemical Journal findings\",\n      \"pmids\": [\"16757333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Rassf1 interacts with Daxx (co-immunoprecipitation) and partially co-localizes with Daxx during mitosis. Rassf1/Daxx depletion or expression of the Daxx-binding domain of Rassf1 elevates cyclin B stability and increases taxol resistance, defining a mitotic stress checkpoint. Rassf1 depletion does not alter APC/C ubiquitin ligase activity in vitro, indicating the checkpoint requires the mitotic cellular environment.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, cyclin B stability assay, in vitro APC/C ubiquitin ligase assay, time-lapse microscopy, mouse xenograft\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus in vitro APC/C assay and in vivo xenograft, single lab\",\n      \"pmids\": [\"21643015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RASSF family members (RASSF1-6) interact with MST kinases through SARAH domain-mediated interactions, confirmed by in vitro interaction studies; RASSF7 was identified as a new MST kinase interacting partner. RA domain interactions show diversity across RASSF members. RASSF proteins act as adaptors assembling multiple protein complexes.\",\n      \"method\": \"In vitro interaction studies (SARAH and RA domain), structural modeling, in silico prediction\",\n      \"journal\": \"Advances in biological regulation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vitro binding assays with domain mapping; single study\",\n      \"pmids\": [\"23357313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RASSF1A restricts SRC kinase activity and prevents motility/invasion, while the RASSF1C isoform (expressed when RASSF1A promoter is methylated) targets SRC/YES kinases to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A promoter methylation reduces YAP phospho-S127, derepressing YAP1; this is identified by affinity proteomics, proximity ligation assay, and real-time molecular visualization.\",\n      \"method\": \"Affinity proteomics, proximity ligation assay, live-cell imaging, phosphorylation assays, in vitro and in vivo invasion/motility assays, promoter methylation analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (proteomics, PLA, imaging, functional assays) in a single study\",\n      \"pmids\": [\"26549256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"miR-193a-3p directly binds to the RASSF1 3'UTR and represses RASSF1 mRNA and protein expression; excess miR-193a-3p impairs the Rassf1-Syntaxin 16 signalling pathway required for cytokinesis completion, causing polyploidy, multipolar mitotic spindles, and elevated cell death.\",\n      \"method\": \"Dual luciferase reporter assay, immunoblotting, qRT-PCR, time-lapse microscopy, immunofluorescence\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — luciferase validation of direct miRNA-3'UTR interaction plus functional cell division phenotype\",\n      \"pmids\": [\"28449010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RASSF1-AS1 (an antisense lncRNA from the RASSF1A locus) partially overlaps RASSF1A mRNA at exon 2 and directly binds RASSF1A mRNA, suppressing its translation at the post-transcriptional level. RNA pulldown and luciferase assays confirm this direct binding. Mutated RASSF1-AS1 lacking the binding region has no effect on RASSF1A expression or NF-κB activation.\",\n      \"method\": \"RNA pulldown, luciferase activity assay, overexpression/knockdown in mouse primary cardiac fibroblasts, Western blot\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNA pulldown plus reporter assay with domain deletion mutant, single lab\",\n      \"pmids\": [\"30571844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"miR-181a directly targets RASSF1 (validated by luciferase assay), reducing RASSF1 expression; knockdown of RASSF1 increases sorafenib resistance in hepatocellular carcinoma cells, linking RASSF1 to MAPK signaling and sorafenib sensitivity.\",\n      \"method\": \"Luciferase reporter assay, miRNA overexpression/inhibition, RNAi knockdown, apoptosis assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — luciferase validation plus functional knockdown, single lab\",\n      \"pmids\": [\"27384977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"rhBMP-2 increases MST1, MOB1, and p-YAP levels in esophageal cancer cells, and RASSF1 binding to MST1 is enhanced upon rhBMP-2 treatment. RASSF1 knockdown reverses the p-YAP induction by rhBMP-2, placing RASSF1 upstream of MST1 in rhBMP-2-activated Hippo signaling.\",\n      \"method\": \"Co-immunoprecipitation (RASSF1-MST1), siRNA knockdown, Western blot, MTT assay, xenograft\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP combined with knockdown rescue in a single study\",\n      \"pmids\": [\"27230238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATF4 directly binds to ATF4-binding sites in the RASSF1 promoter (shown by ChIP) and activates RASSF1 transcription (shown by reporter assay). RASSF1 ablation mitigates expression of the ATF4 effector BBC3 and abrogates tunicamycin-induced apoptosis, placing RASSF1 downstream of ATF4 in ER stress-associated apoptosis.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), luciferase reporter assay, RASSF1 knockdown, tunicamycin-induced apoptosis assay\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus reporter assay plus functional KO phenotype, single lab\",\n      \"pmids\": [\"36723232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"P. multocida infection upregulates Rassf1 expression in pulmonary epithelial cells; Rassf1 enhances Hippo-YAP pathway through phosphorylation (increased p-MST1/2, p-LATS1, p-YAP). In vitro knockdown of Rassf1 enhances YAP activity and reduces apoptosis during infection, and pharmacological Hippo inhibition rescues apoptosis in vitro and reduces lung injury in vivo.\",\n      \"method\": \"RNA-seq, siRNA knockdown, Western blot for pathway phosphorylation, mouse and rabbit infection models, pharmacological inhibition (XMU-MP-1)\",\n      \"journal\": \"Veterinary research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro KD with defined phosphorylation readout plus in vivo model; single lab\",\n      \"pmids\": [\"38493147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFX2 transcription factor binds directly to the RASSF1 promoter (shown by dual-luciferase and ChIP assays) and activates RASSF1 transcription. RASSF1 knockdown reverses the ability of RFX2 overexpression to inhibit immune escape, placing RASSF1 downstream of RFX2 in Hippo/YAP-mediated immune regulation.\",\n      \"method\": \"Dual-luciferase reporter assay, ChIP-PCR, siRNA knockdown, co-culture with CD8+ T cells, Western blot for YAP phosphorylation\",\n      \"journal\": \"Cell division\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus reporter assay with functional rescue, single lab\",\n      \"pmids\": [\"40069841\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RASSF1A functions as a scaffold/adaptor tumor suppressor that binds Ras-GTP through its RA domain and MST1/2 kinases through its SARAH domain; it inhibits MST1/2 autoactivation in quiescent cells while directing them to sites of activation, stabilizes microtubules, restricts SRC kinase activity at cell-cell junctions, and promotes apoptosis downstream of oncogenic Ras and ER stress (ATF4), whereas the alternative RASSF1C isoform has opposing pro-tumorigenic effects including SRC/YES activation and YAP1 derepression upon epigenetic silencing of RASSF1A.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RASSF1 is a Ras effector and scaffold protein that integrates Ras-GTP signaling, Hippo pathway regulation, and microtubule dynamics to exert tumor-suppressive functions. It binds Ras in a GTP-dependent manner through its RA domain and mediates caspase-dependent apoptosis downstream of activated Ras, ATF4-driven ER stress, and BMP-2 signaling [PMID:10998413, PMID:36723232, PMID:27230238]. Through its SARAH domain, RASSF1A interacts with MST1/2 kinases, restraining their autoactivation in quiescent cells while facilitating context-dependent Hippo pathway activation that promotes YAP phosphorylation and suppresses proliferation [PMID:15109305, PMID:16757333, PMID:38493147]. RASSF1A additionally stabilizes microtubules, cooperates with Daxx in a mitotic checkpoint controlling cyclin B turnover, and restricts SRC kinase activity at cell–cell junctions; loss of RASSF1A by promoter methylation shifts signaling toward the RASSF1C isoform, which activates SRC/YES and derepresses YAP1 to promote invasion [PMID:16135822, PMID:21643015, PMID:26549256].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing RASSF1 as a direct Ras effector answered the question of whether RAS-family GTPases signal through RASSF proteins and showed that RASSF1-mediated apoptosis is Ras-dependent and caspase-dependent.\",\n      \"evidence\": \"In vitro pulldown, co-IP, and epistasis with dominant-active/dominant-negative Ras in transfected cells\",\n      \"pmids\": [\"10998413\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of GTP-dependent Ras–RASSF1 interaction not determined\",\n        \"Endogenous Ras–RASSF1 complex not demonstrated\",\n        \"Downstream caspase cascade not mapped\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identification of C19ORF5 as a RASSF1 interactor and visualization of RASSF1 on microtubules and mitochondria revealed that RASSF1 operates at the cytoskeleton and mitochondria, not solely in cytosolic Ras signaling.\",\n      \"evidence\": \"Yeast two-hybrid screen and GFP co-localization with β-tubulin and mitochondria\",\n      \"pmids\": [\"12762840\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional significance of mitochondrial localization unclear\",\n        \"Direct binding to tubulin not demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstration that RASSF1A inhibits MST1 autoactivation in vitro yet permits activation when recruited to membranes established the dual-function scaffold model: RASSF1A restrains MST1/2 in the cytoplasm but enables context-dependent activation at membranes.\",\n      \"evidence\": \"Endogenous co-IP from KB cells, reconstituted in vitro kinase assays with purified proteins, CAAX-targeted constructs\",\n      \"pmids\": [\"15109305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vivo physiological membrane-targeting signal unknown\",\n        \"Stoichiometry of RASSF1A–MST1 complex not quantified\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery of the RASSF1–PMCA4b interaction at the plasma membrane, which inhibits EGF-driven ERK signaling, revealed a non-Ras mechanism by which RASSF1 limits MAPK activation.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, GST pulldown, and ERK activity rescue with competing peptide\",\n      \"pmids\": [\"15145946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether PMCA4b–RASSF1 interaction is isoform-specific (RASSF1A vs 1C) not resolved\",\n        \"In vivo relevance in animal models not tested\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Rassf1A knockout mice showed increased tumorigenesis and MEF hypersensitivity to microtubule-destabilizing agents, providing genetic proof that RASSF1A functions as a tumor suppressor through microtubule stabilization in vivo.\",\n      \"evidence\": \"Targeted gene deletion in mouse, primary fibroblast drug sensitivity assays, tumor monitoring\",\n      \"pmids\": [\"16135822\", \"15753381\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether microtubule stabilization or Hippo regulation is the primary tumor-suppressive mechanism not distinguished\",\n        \"Tissue-specific contributions not dissected\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of the RASSF1–Daxx interaction and its role in a mitotic checkpoint that regulates cyclin B stability linked RASSF1 to mitotic fidelity beyond simple microtubule stabilization.\",\n      \"evidence\": \"Co-IP, RNAi, time-lapse microscopy, cyclin B stability assays, in vitro APC/C assay\",\n      \"pmids\": [\"21643015\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which RASSF1–Daxx promotes cyclin B degradation without altering APC/C activity in vitro not explained\",\n        \"Single-lab finding; independent replication absent\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Affinity proteomics and proximity ligation revealed that RASSF1A restricts SRC activity while RASSF1C activates SRC/YES at cell junctions, explaining how promoter methylation-driven isoform switching derepresses YAP1 and promotes invasion.\",\n      \"evidence\": \"Affinity proteomics, PLA, live-cell imaging, phosphorylation assays in epithelial cells\",\n      \"pmids\": [\"26549256\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct binding interface between RASSF1C and SRC/YES not mapped\",\n        \"Whether RASSF1C activates YAP independently of MST1/2 not resolved\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placing RASSF1 upstream of MST1–YAP in BMP-2 signaling in esophageal cancer cells extended the RASSF1–Hippo axis to growth factor contexts beyond canonical Ras.\",\n      \"evidence\": \"Co-IP of RASSF1–MST1 under BMP-2 treatment, siRNA knockdown, xenograft\",\n      \"pmids\": [\"27230238\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single co-IP without reciprocal validation under BMP-2\",\n        \"Whether BMP-2 triggers RASSF1A membrane recruitment not tested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"ChIP and reporter assays showing ATF4 directly activates RASSF1 transcription, and that RASSF1 loss abrogates tunicamycin-induced apoptosis, established RASSF1 as a transcriptional target of the integrated stress response mediating ER stress-induced cell death.\",\n      \"evidence\": \"ChIP, luciferase reporter, RASSF1 knockdown, tunicamycin-induced apoptosis\",\n      \"pmids\": [\"36723232\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether RASSF1A or RASSF1C is the isoform induced by ATF4 not specified\",\n        \"Downstream effector pathway (Hippo vs other) linking RASSF1 to BBC3 not resolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstration that Rassf1 activates the full Hippo phosphorylation cascade (p-MST1/2 → p-LATS1 → p-YAP) during bacterial infection-induced apoptosis in pulmonary epithelial cells extended RASSF1–Hippo signaling to innate immune and infection contexts.\",\n      \"evidence\": \"RNA-seq, siRNA knockdown, phospho-Western blots, mouse/rabbit infection models, XMU-MP-1 rescue\",\n      \"pmids\": [\"38493147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct physical interaction between RASSF1 and MST1/2 not confirmed in pulmonary epithelial cells\",\n        \"Whether RASSF1 acts cell-autonomously or through paracrine signaling not distinguished\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural basis for RASSF1A's dual role as MST1/2 inhibitor in the cytoplasm versus activator at membranes; the relative contributions of microtubule stabilization, Hippo activation, and SRC restriction to tumor suppression; and the mechanism by which RASSF1C opposes RASSF1A function at the molecular level.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No high-resolution structure of RASSF1A–MST1 SARAH domain complex\",\n        \"Genetic separation of microtubule-stabilizing vs Hippo-activating functions not achieved in vivo\",\n        \"Biochemical basis by which RASSF1C activates SRC/YES while RASSF1A inhibits it not defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 6, 9, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 6, 9, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 14, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 6, 9, 13, 15]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MST1\",\n      \"MST2\",\n      \"DAXX\",\n      \"ATP2B4\",\n      \"SRC\",\n      \"YES1\",\n      \"C19orf5\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}