{"gene":"SF3B4","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":1997,"finding":"C. elegans SAP49 (SF3B4 ortholog) possesses specific RNA-binding activity residing in its second RNA recognition motif (RRM2), as demonstrated by in vitro RNA selection (SELEX).","method":"In vitro RNA selection (SELEX), RNA-binding assay","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assay with domain mapping, single lab, single organism ortholog","pmids":["9163526"],"is_preprint":false},{"year":1998,"finding":"SAP49 (SF3B4) cross-linking to retroviral pre-mRNA is enhanced at step 2 of splicing when spliceosomal progression is blocked, indicating SF3B4 regulates late-stage spliceosome transitions; UV cross-linking showed altered SAP49 binding correlates with defective transit through the last spliceosomal complex.","method":"UV cross-linking of cellular splicing factors to viral RNA substrates, spliceosomal complex analysis in cell-free splicing system","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro cell-free splicing assay with cross-linking, single lab, single study","pmids":["9614130"],"is_preprint":false},{"year":2016,"finding":"The solution structure of the first RRM domain (RRM1) of human SF3B4 (SF3b49) was solved by NMR, revealing a canonical β1-α1-β2-β3-α2-β4 fold. RRM1 interacts with residues 607-616 of SF3B145 via a unique antiparallel helix-helix interaction involving SF3B4 α1; this interaction was confirmed by GST pull-down and mutational analysis.","method":"NMR structure determination, chemical shift mapping, NOESY-based docking, GST pull-down with mutational analysis","journal":"Protein science : a publication of the Protein Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure solved, interaction mapped by chemical shift and NOESY, validated by mutagenesis and GST pull-down in one rigorous study","pmids":["27862552"],"is_preprint":false},{"year":2016,"finding":"Knockdown of Sf3b4 in Xenopus laevis embryos reduces expression of neural crest specifier genes (sox10, snail2, twist) at the neural plate border and decreases neural crest cells in pharyngeal arches, resulting in hypoplasia of neural crest-derived craniofacial cartilages. This phenotype is rescued by wild-type human SF3B4 mRNA but not by Nager syndrome mutant mRNAs, establishing a direct requirement for SF3B4 function in neural crest development.","method":"Morpholino antisense knockdown in Xenopus laevis, in situ hybridization, mRNA rescue with wild-type vs. mutant SF3B4","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — morpholino loss-of-function with defined phenotypic readout, mRNA rescue with mutant vs. wild-type discrimination, replicated across developmental stages","pmids":["26874011"],"is_preprint":false},{"year":2018,"finding":"SF3B4 knockdown in liver cancer cells causes G1/S cell cycle arrest by upregulating p27Kip1 and suppressing cyclins and CDKs. SF3B4 overexpression causes aberrant splicing of the KLF4 tumor suppressor pre-mRNA, generating non-functional exon-skipped transcripts, which leads to transcriptional inactivation of p27Kip1 and activation of Slug (SNAI2).","method":"siRNA knockdown, western blotting, RT-PCR for alternative splicing, cell cycle analysis","journal":"BMB reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with defined cell cycle phenotype and splicing mechanism identified, single lab","pmids":["29397868"],"is_preprint":false},{"year":2018,"finding":"miR-133b directly targets and negatively regulates SF3B4 expression; miR-133b mimics partially abolish the effects of SF3B4 overexpression in HCC cells, and SF3B4 downstream molecules KLF4, KIP1, and SNAI2 are also modulated by miR-133b.","method":"TargetScan prediction, miRNA mimic transfection, western blotting, in vitro functional assays, xenograft mouse model","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — miRNA-target relationship confirmed by functional rescue, single lab","pmids":["30391496"],"is_preprint":false},{"year":2020,"finding":"Heterozygous knockout of Sf3b4 in mice causes homeotic posteriorization of vertebral morphology, flattened calvaria, and reduced cell proliferation in the forebrain, with homozygous deletion being embryonic lethal; this establishes Sf3b4 as required for axial skeletal patterning and forebrain development in vivo.","method":"CRISPR-Cas9 knockout mouse generation, phenotypic analysis of heterozygous and homozygous mutants, histology","journal":"Developmental dynamics : an official publication of the American Association of Anatomists","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo genetic knockout with multiple defined phenotypic readouts, homozygous lethality and heterozygous defects characterized","pmids":["31900962"],"is_preprint":false},{"year":2020,"finding":"SRSF3 knockdown significantly increases SF3B4 mRNA and protein levels in HCC cells, likely by retarding SF3B4 mRNA decay rates; the coding region of SF3B4 is involved in SRSF3-mediated regulation of SF3B4 expression.","method":"siRNA knockdown, RT-qPCR, western blotting, GFP-SF3B4 fusion construct analysis","journal":"Anticancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, single method (KD with mRNA level measurement), mechanism (mRNA stability) inferred but not directly demonstrated","pmids":["32234894"],"is_preprint":false},{"year":2022,"finding":"SF3B4 knockdown in A549 NSCLC cells causes G0/G1 arrest, upregulation of p27, p21, and p53. SF3B4 interacts with UBE4B (ubiquitination factor E4B) by co-immunoprecipitation; SF3B4 depletion reduces UBE4B levels, which in turn reduces polyubiquitinated p53, leading to p53 accumulation and p21/p27 induction. Double knockdown of SF3B4 and p53 partially restores p21 expression and cell proliferation.","method":"siRNA knockdown, co-immunoprecipitation, western blotting, flow cytometry, double-knockdown epistasis","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reciprocal co-IP identifying SF3B4-UBE4B interaction, epistasis by double knockdown, single lab","pmids":["35996826"],"is_preprint":false},{"year":2022,"finding":"miR-509-3p binds the 3'-UTR of SF3B4 mRNA to decrease its expression. SF3B4 promotes effective splicing of RAD52 pre-mRNA; SF3B4 loss causes intron 8 retention in RAD52 and generation of premature termination codons, reducing RAD52 protein. Decreased RAD52 partially counteracts the tumor-promoting effects of SF3B4 overexpression in ovarian cancer cells.","method":"3'-UTR luciferase reporter assay, RNA-seq/alternative splicing analysis, siRNA knockdown, in vitro and in vivo functional assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — splicing mechanism identified by RNA-seq and validated functionally by rescue experiment, single lab","pmids":["35210412"],"is_preprint":false},{"year":2022,"finding":"SF3B4 promotes effective splicing of SPAG5 pre-mRNA in cervical cancer cells; SF3B4 knockdown causes intron retention and reduced maturation of SPAG5 pre-mRNA. SPAG5 deficiency impairs the oncogenic effects of SF3B4 overexpression.","method":"RNA-seq and alternative splicing analysis, siRNA knockdown, rescue experiments, in vitro and in vivo assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — splicing mechanism identified by RNA-seq and validated functionally, single lab","pmids":["35853859"],"is_preprint":false},{"year":2022,"finding":"SF3B4 binds ENAH mRNA and stabilizes it (as shown by RIP and actinomycin D mRNA decay assay), leading to elevated ENAH protein which activates Notch signaling to promote HCC cell proliferation, invasion, and migration.","method":"RNA immunoprecipitation (RIP), actinomycin D mRNA stability assay, western blotting, functional cell assays","journal":"Bioengineered","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — RIP and mRNA stability assay establishing SF3B4-ENAH mRNA binding, single lab","pmids":["35030977"],"is_preprint":false},{"year":2022,"finding":"SF3B4 binds GPAA1 mRNA (confirmed by RNA immunoprecipitation), and SF3B4 overexpression reverses the effects of GPAA1 knockdown on HCC cell proliferation, migration, and invasion.","method":"RNA immunoprecipitation (RIP), siRNA knockdown, rescue overexpression, functional assays","journal":"Oncology letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single RIP experiment identifying RNA binding, functional link by rescue, single lab","pmids":["35399327"],"is_preprint":false},{"year":2022,"finding":"SF3B4 binds the DR8 intronic cis-element of hTERT pre-mRNA (demonstrated by RNA immunoprecipitation in NSCLC cells); SF3B4 knockdown reduces full-length hTERT splicing, telomerase activity, telomere length, and cancer cell growth, establishing SF3B4 as a trans-acting splicing factor for hTERT in lung cancer.","method":"RNA immunoprecipitation (RIP), RT-PCR for hTERT isoforms, telomerase activity assay, telomere length measurement, siRNA knockdown","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — RIP demonstrating direct binding to hTERT pre-mRNA, functional consequences measured by multiple assays, single lab","pmids":["35852380"],"is_preprint":false},{"year":2023,"finding":"SF3B4 promotes KLF16 mRNA export from nucleus to cytoplasm, thereby increasing KLF16 protein expression; elevated KLF16 directly binds the Twist1 promoter to activate its transcription, driving EMT and ccRCC progression.","method":"siRNA knockdown, nuclear/cytoplasmic fractionation, western blotting, chromatin immunoprecipitation (ChIP), functional migration/invasion assays, in vivo xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — fractionation demonstrating mRNA export function, ChIP for KLF16-Twist1 promoter binding, single lab","pmids":["36639679"],"is_preprint":false},{"year":2023,"finding":"Heterozygous Sf3b4 knockout mice exhibit abnormal vertebral development accompanied by changes in Hox gene expression levels and patterns in somites; RNA-seq reveals widespread differential splicing events (predominantly exon skipping and intron retention) in chromatin remodeler transcripts known to regulate Hox expression, suggesting Sf3b4 controls Hox-dependent vertebral patterning via splicing of chromatin remodelers.","method":"CRISPR/Cas9 heterozygous knockout mice, RNA-seq of whole embryos and somites, in situ hybridization for Hox genes","journal":"Differentiation; research in biological diversity","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo genetic model with RNA-seq identifying splicing targets, pathway placement by transcriptomic analysis, single lab","pmids":["37167859"],"is_preprint":false},{"year":2023,"finding":"sf3b4-null zebrafish display retinitis pigmentosa-like phenotypes including retinal pigment epithelium defects and rod degeneration; transcriptome analysis reveals altered retinol metabolism and retinoic acid signaling, and retinoic acid supplementation rescues key cellular phenotypes in sf3b4-/- mutants.","method":"sf3b4-/- zebrafish mutants, transcriptome profiling, immunostaining, behavioral analysis, retinoic acid rescue","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic knockout with defined retinal phenotype, pathway rescue by retinoic acid, single lab","pmids":["37263342"],"is_preprint":false},{"year":2024,"finding":"Conditional homozygous deletion of Sf3b4 in neural crest cells (NCC) in mice causes craniofacial and cardiac malformations; RNA-seq prior to morphological defects reveals exon skipping in NCC regulatory network genes and in histone modifier transcripts, with enrichment of thymidine bases around branch points of affected exons, suggesting Sf3b4 regulates branchpoint-proximal splicing of NCC and cardiac developmental genes.","method":"NCC-specific conditional knockout mice, RNA-seq, splicing analysis, branchpoint sequence analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic model with tissue-specific deletion, mechanistic RNA-seq establishing splicing targets, branchpoint enrichment analysis, replicated craniofacial phenotype across models","pmids":["39292749"],"is_preprint":false},{"year":2024,"finding":"SF3B4 knockdown in LUAD cells causes an alternative 5'-splice site in KAT2A pre-mRNA, reducing KAT2A at RNA and protein levels; decreased KAT2A partially reverses SF3B4-dependent LUAD growth promotion, establishing the SF3B4/KAT2A axis in lung adenocarcinoma.","method":"siRNA knockdown, RT-PCR for alternative splicing, western blotting, rescue overexpression, functional proliferation/migration assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — alternative splicing event identified and functionally validated by KAT2A rescue, single lab","pmids":["38168564"],"is_preprint":false},{"year":2024,"finding":"METTL3 promotes SF3B4 mRNA stability through m6A modification (confirmed by MeRIP assay); SF3B4 interacts with LSM4 (confirmed by co-IP); SF3B4 depletion suppresses NSCLC cell functions that are rescued by LSM4 overexpression, placing SF3B4 upstream of LSM4 in a METTL3-SF3B4-LSM4 regulatory axis.","method":"MeRIP (m6A RIP), co-immunoprecipitation, siRNA knockdown, rescue overexpression, xenograft mouse model","journal":"Thoracic cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — MeRIP for m6A modification, Co-IP for SF3B4-LSM4 interaction, epistasis by rescue, single lab","pmids":["38462740"],"is_preprint":false},{"year":2024,"finding":"SF3B4 deficiency leads to dissociation of NMD factors UPF1, MAGOH, and RNPS1 from the 3'UTR of p21 mRNA, thereby stabilizing p21 mRNA and inducing senescence via p53-independent p21 upregulation; SF3B4 recruits these NMD factors to facilitate p21 mRNA decay.","method":"siRNA knockdown, SA-β-Gal staining, RT-qPCR, western blotting, RNA immunoprecipitation for NMD factor-mRNA association","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — RIP demonstrating SF3B4-NMD factor-p21 mRNA complex, functional senescence readout, single lab","pmids":["39961431"],"is_preprint":false},{"year":2024,"finding":"SETDB1 acts as a transcription factor that binds the SF3B4 promoter (confirmed by dual-luciferase reporter assay) to activate SF3B4 expression; SF3B4 knockdown impairs SETDB1-driven ovarian cancer cell proliferation and motility.","method":"Cistrome DB analysis, dual-luciferase reporter assay, siRNA knockdown, functional cell assays","journal":"Journal of ovarian research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — luciferase reporter confirming SETDB1-SF3B4 promoter interaction, epistasis by double knockdown, single lab","pmids":["38317200"],"is_preprint":false},{"year":2025,"finding":"Homozygous deletion of sf3b4 in Xenopus tropicalis causes increased exon-skipping events followed by transcriptional changes affecting cranial neural crest cell migration and survival; loss of one sf3b4 copy is largely inconsequential, but homozygous loss causes major splicing defects disrupting NC-derived craniofacial cartilage.","method":"CRISPR/Cas9 sf3b4 knockout in Xenopus tropicalis, temporal RNA-seq, phenotypic characterization","journal":"Disease models & mechanisms","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic model with temporal RNA-seq, replicated across Xenopus species and consistent with mouse NCC knockout","pmids":["40126363"],"is_preprint":false},{"year":2025,"finding":"SF3B4 siRNA knockdown in human embryonic stem cell-derived neural crest differentiation reduces neural crest gene expression, increases apoptosis, and causes premature neuronal differentiation, establishing a cell-autonomous requirement for SF3B4 in human neural crest cell survival, maintenance, and differentiation.","method":"siRNA knockdown in hESC-derived neural crest differentiation, qRT-PCR for NC markers, apoptosis assays","journal":"Developmental dynamics : an official publication of the American Association of Anatomists","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — human stem cell model with defined NC differentiation phenotype, single lab","pmids":["40047147"],"is_preprint":false},{"year":2025,"finding":"SF3B4 interacts with BUB1 (checkpoint protein) and regulates cancer cell mitosis and proliferation in HCC; identified by genome-wide CRISPR KO screening in patient-derived HCC organoids as a top essential survival gene.","method":"Genome-wide CRISPR knockout screen in HCC organoids, RNA immunoprecipitation-seq, long-read isoform sequencing, RNA-seq, hydrodynamic tail vein injection HCC model","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen in organoid model, multiple orthogonal transcriptomic methods, in vivo validation, single lab","pmids":["41071884"],"is_preprint":false},{"year":2025,"finding":"SF3B4 regulates alternative splicing of GLS1 pre-mRNA, preferentially promoting production of the high-activity GAC isoform over the KGA isoform; SF3B4 inhibition reduces GAC expression, decreases GLS enzymatic activity, impairs glutaminolysis, and suppresses glutamine-driven mitochondrial respiration in HCC cells.","method":"siRNA/shRNA knockdown, RT-PCR for GLS1 isoforms, GLS enzymatic activity assay, metabolic flux analysis, functional cell assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — isoform splicing identified and enzymatic activity measured, metabolic phenotype characterized, single lab","pmids":["41411906"],"is_preprint":false},{"year":2025,"finding":"SF3B4 directly binds the 3'UTR of SREBF1 mRNA (confirmed by RIP, RNA pull-down, and luciferase reporter), enhancing SREBF1 mRNA stability; SREBF1 overexpression partially rescues malignant phenotypes suppressed by SF3B4 knockdown in HCC.","method":"RNA immunoprecipitation, RNA pull-down, dual-luciferase reporter assay, mRNA stability assay, Oxford Nanopore transcriptome sequencing, xenograft model","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple orthogonal methods confirming direct 3'UTR binding and mRNA stabilization, functional rescue, single lab","pmids":["42255207"],"is_preprint":false},{"year":2025,"finding":"EP300 and CREBBP activate SF3B4 expression by promoting H3K27ac modification on the SF3B4 promoter; SF3B4 knockdown-mediated inhibition of CRC cell proliferation is counteracted by enhanced autophagy.","method":"ChIP for H3K27ac at SF3B4 promoter, siRNA knockdown, autophagy assays, functional proliferation assays","journal":"American journal of cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP for histone modification at SF3B4 promoter, functional link through autophagy inferred, single lab","pmids":["40667544"],"is_preprint":false},{"year":2025,"finding":"SF3B4 promotes melanoma cell migration through splicing-dependent regulation of Talin1; SF3B4 knockdown reduces Talin1 mRNA and protein levels, causes Talin1 intron retention, and reduces FAK phosphorylation; combined SF3B4+Talin1 knockdown shows no additive migration defect, placing them in the same pathway.","method":"siRNA knockdown, RT-qPCR, western blotting for Talin1 and phospho-FAK, wound healing and transwell migration assays, double knockdown epistasis","journal":"Cancer genomics & proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — splicing mechanism identified, epistasis by double knockdown supporting shared pathway, single lab","pmids":["41771579"],"is_preprint":false},{"year":2025,"finding":"SF3B4 loss triggers skipping of exon 2 in CCND1 pre-mRNA, preventing formation of a CCND1-CDK6 holoenzyme complex and blocking the G1-S transition; rescue with a CCND1 isoform containing the skipped exon restores both CDK6 complex formation and cell proliferation.","method":"CRISPore-seq (CRISPR perturbation + long-read single-cell transcriptomics), co-immunoprecipitation for CCND1-CDK6 complex, isoform rescue experiment","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — isoform-level sequencing identifying specific exon skipping, co-IP for complex formation, rescue confirming mechanism, single lab preprint","pmids":["bio_10.1101_2025.11.25.690515"],"is_preprint":true},{"year":2025,"finding":"SF3B4 depletion upregulates natural NMD-targeted mRNA isoforms; among spliceosome components tested, SF3B4 (along with AQR, SF3B1, and CDC40) may have a more direct role in NMD beyond indirect effects from widespread splicing disruption.","method":"Analysis of publicly available RNA-seq datasets from cells depleted of 18 spliceosome components (K562 and HepG2)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational re-analysis of existing RNA-seq datasets, no direct biochemical experiment on SF3B4's NMD role, preprint","pmids":["bio_10.1101_2024.12.27.630533"],"is_preprint":true},{"year":2025,"finding":"SF3B4 regulates HCC cell proliferation and apoptosis through alternative splicing; iRIP-seq identifies 252 direct RNA targets; SF3B4 may bind TRIM28 mRNA to modulate its expression and interact with SETD5 to influence pre-mRNA alternative splicing.","method":"iRIP-seq, RNA-seq after SF3B4 knockdown, co-immunoprecipitation","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — iRIP-seq identifying direct RNA targets with transcriptome-wide splicing analysis, single lab","pmids":["40234915"],"is_preprint":false}],"current_model":"SF3B4 (SAP49/SF3b49) is a core subunit of the SF3b complex within the U2 snRNP whose RRM1 domain adopts a canonical fold and engages SF3B145 via a unique antiparallel helix-helix interaction; it functions at the branchpoint recognition step of pre-mRNA splicing—with loss causing predominantly exon skipping and intron retention across hundreds of transcripts—and beyond canonical splicing it stabilizes specific mRNAs via 3'UTR binding (e.g., SREBF1, ENAH, GPAA1), promotes mRNA nuclear export (KLF16), recruits NMD factors (UPF1, MAGOH, RNPS1) to target p21 mRNA for decay, and controls isoform switching of GLS1, CCND1, RAD52, SPAG5, KAT2A, and hTERT pre-mRNAs; in neural crest cells SF3B4 is cell-autonomously required for survival, migration, and differentiation, and its haploinsufficiency causes the craniofacial and cardiac defects of Nager/Rodriguez syndrome."},"narrative":{"mechanistic_narrative":"SF3B4 (SAP49/SF3b49) is a core RNA-binding subunit of the SF3b complex within the U2 snRNP that acts at the branchpoint recognition step of pre-mRNA splicing, where its loss produces predominantly exon skipping and intron retention across developmental and cancer transcript networks [PMID:39292749, PMID:40126363]. The protein contains tandem RNA recognition motifs—RRM2 carries sequence-specific RNA-binding activity, while the canonically folded RRM1 docks onto SF3B145 (SF3B3) through a unique antiparallel helix-helix interaction that anchors SF3B4 within the SF3b complex [PMID:9163526, PMID:27862552]. In vivo, SF3B4 governs neural crest cell survival, migration, and differentiation cell-autonomously, and its loss disrupts craniofacial cartilage formation, axial vertebral patterning through Hox-regulating chromatin remodeler splicing, and forebrain development [PMID:26874011, PMID:31900962, PMID:37167859, PMID:39292749, PMID:40126363, PMID:40047147]; haploinsufficiency underlies the craniofacial defects modeled by Nager-syndrome mutant mRNAs that fail to rescue SF3B4 loss [PMID:26874011]. Beyond canonical splicing, SF3B4 controls isoform switching of multiple cancer-relevant pre-mRNAs—generating high-activity GLS1 GAC, full-length hTERT, exon-2-containing CCND1, and functional RAD52, SPAG5, KAT2A, and KLF4 transcripts [PMID:29397868, PMID:35210412, PMID:35853859, PMID:35852380, PMID:38168564, PMID:41411906, PMID:bio_10.1101_2025.11.25.690515]—and exerts post-splicing control by stabilizing target mRNAs through 3'UTR binding (SREBF1, ENAH), promoting nuclear export of KLF16, and recruiting the NMD factors UPF1, MAGOH, and RNPS1 to drive p21 mRNA decay, thereby restraining senescence [PMID:35030977, PMID:36639679, PMID:39961431, PMID:42255207]. Through these activities SF3B4 promotes proliferation and cell-cycle progression, with depletion causing G1/S arrest via p27/p21/p53 induction, in part through interaction with UBE4B [PMID:29397868, PMID:35996826].","teleology":[{"year":1997,"claim":"Established that the SF3B4 ortholog is a genuine sequence-specific RNA-binding protein and localized that activity to a defined domain, framing it as a direct RNA-engaging splicing factor rather than a passive scaffold.","evidence":"In vitro RNA selection (SELEX) on C. elegans SAP49, with domain mapping to RRM2","pmids":["9163526"],"confidence":"Medium","gaps":["RNA target identity in human SF3B4 not defined","single ortholog, single in vitro assay"]},{"year":1998,"claim":"Linked SF3B4 dynamics to spliceosomal progression, showing its binding changes at the catalytic transitions of splicing.","evidence":"UV cross-linking of SAP49 to retroviral pre-mRNA in a cell-free splicing system under spliceosomal stalling","pmids":["9614130"],"confidence":"Medium","gaps":["mechanism of altered binding at step 2 not resolved","single substrate, single study"]},{"year":2016,"claim":"Resolved how SF3B4 is structurally integrated into the SF3b complex by solving its RRM1 fold and the molecular interface with SF3B145.","evidence":"NMR structure of human SF3B4 RRM1, chemical shift mapping, NOESY docking, GST pull-down with mutagenesis","pmids":["27862552"],"confidence":"High","gaps":["functional consequence of RRM1-SF3B145 interaction for splicing not tested in vivo","RRM2 RNA-binding role not structurally addressed here"]},{"year":2016,"claim":"Demonstrated a direct developmental requirement for SF3B4 in neural crest specification and connected Nager-syndrome mutations to functional loss via rescue discrimination.","evidence":"Morpholino knockdown in Xenopus laevis with in situ hybridization and wild-type vs. mutant mRNA rescue","pmids":["26874011"],"confidence":"High","gaps":["splicing targets mediating the phenotype not identified","morpholino specificity relies on rescue alone"]},{"year":2018,"claim":"Connected SF3B4 to cell-cycle control and tumor biology, identifying KLF4 mis-splicing as a route to p27/Slug dysregulation.","evidence":"siRNA knockdown and overexpression in liver cancer cells, RT-PCR splicing analysis, cell cycle profiling","pmids":["29397868"],"confidence":"Medium","gaps":["direct SF3B4 binding to KLF4 pre-mRNA not shown","single cancer cell context"]},{"year":2018,"claim":"Identified upstream control of SF3B4 by miR-133b, framing SF3B4 levels as a regulated node in HCC.","evidence":"TargetScan prediction, miRNA mimic transfection, functional and xenograft assays","pmids":["30391496"],"confidence":"Medium","gaps":["direct miRNA-SF3B4 binding inferred from prediction plus rescue","single lab"]},{"year":2020,"claim":"Established the in vivo requirement of Sf3b4 for axial skeletal patterning and forebrain development through a mammalian genetic model, with homozygous loss being lethal.","evidence":"CRISPR-Cas9 Sf3b4 knockout mice, phenotypic and histological analysis","pmids":["31900962"],"confidence":"High","gaps":["molecular splicing targets not defined in this study","dosage-sensitivity mechanism unresolved"]},{"year":2020,"claim":"Added a layer of expression control by linking SRSF3 to SF3B4 mRNA stability.","evidence":"siRNA knockdown, RT-qPCR, western blotting, GFP-SF3B4 construct analysis in HCC","pmids":["32234894"],"confidence":"Medium","gaps":["mRNA decay mechanism inferred not directly measured","single method, single lab"]},{"year":2022,"claim":"Defined a protein-level mechanism by which SF3B4 restrains p53, showing SF3B4-UBE4B interaction sustains p53 polyubiquitination and thereby proliferation.","evidence":"Reciprocal co-IP, double-knockdown epistasis, flow cytometry in A549 NSCLC cells","pmids":["35996826"],"confidence":"Medium","gaps":["whether interaction is direct or splicing-independent unclear","single lab"]},{"year":2022,"claim":"Built a recurring paradigm of SF3B4-dependent productive splicing of oncogenic targets, here for RAD52 and SPAG5, where loss causes intron retention and PTC generation.","evidence":"3'UTR luciferase, RNA-seq splicing analysis, knockdown and functional rescue in ovarian and cervical cancer cells","pmids":["35210412","35853859"],"confidence":"Medium","gaps":["direct SF3B4 binding to these pre-mRNAs not always shown","single lab per target"]},{"year":2022,"claim":"Extended SF3B4 function beyond splicing into mRNA stabilization via 3'UTR binding, establishing a post-transcriptional regulatory mode (ENAH, GPAA1).","evidence":"RNA immunoprecipitation, actinomycin D decay assays, rescue functional assays in HCC","pmids":["35030977","35399327"],"confidence":"Medium","gaps":["how a spliceosomal factor stabilizes cytoplasmic mRNA mechanistically unclear","GPAA1 link rests on single RIP plus rescue"]},{"year":2022,"claim":"Showed SF3B4 acts as a trans-acting splicing factor for hTERT via a defined intronic cis-element, coupling it to telomerase activity.","evidence":"RIP to the DR8 element, hTERT isoform RT-PCR, telomerase and telomere assays in NSCLC","pmids":["35852380"],"confidence":"Medium","gaps":["structural basis of DR8 recognition unknown","single lab"]},{"year":2023,"claim":"Revealed an mRNA nuclear-export function for SF3B4, driving KLF16-Twist1-mediated EMT.","evidence":"Nuclear/cytoplasmic fractionation, ChIP for KLF16-Twist1, xenograft in ccRCC","pmids":["36639679"],"confidence":"Medium","gaps":["export machinery SF3B4 engages not identified","directness of KLF16 mRNA binding not shown"]},{"year":2023,"claim":"Connected the in vivo skeletal phenotype to a splicing mechanism, implicating mis-splicing of chromatin remodelers that regulate Hox genes.","evidence":"Heterozygous Sf3b4 knockout mice, RNA-seq of somites, Hox in situ hybridization","pmids":["37167859"],"confidence":"Medium","gaps":["causal chromatin-remodeler target not pinpointed","correlative transcriptomic placement"]},{"year":2023,"claim":"Broadened the in vivo phenotypic spectrum to retinal degeneration with a metabolic-pathway link, rescued by retinoic acid.","evidence":"sf3b4-null zebrafish, transcriptomics, retinoic acid rescue","pmids":["37263342"],"confidence":"Medium","gaps":["splicing targets driving retinol metabolism changes not defined","single model"]},{"year":2024,"claim":"Provided the strongest mechanistic placement of SF3B4 at branchpoint-proximal splicing in vivo, tying neural crest and cardiac developmental gene networks to exon skipping.","evidence":"NCC-specific conditional knockout mice, RNA-seq, branchpoint sequence enrichment analysis","pmids":["39292749"],"confidence":"High","gaps":["individual causal target exons among NCC genes not isolated","thymidine/branchpoint enrichment correlative"]},{"year":2024,"claim":"Added KAT2A isoform control to the SF3B4 splicing-target repertoire in lung adenocarcinoma.","evidence":"siRNA knockdown, alternative 5'-splice-site RT-PCR, functional rescue","pmids":["38168564"],"confidence":"Medium","gaps":["direct pre-mRNA binding not demonstrated","single lab"]},{"year":2024,"claim":"Layered m6A-dependent stabilization (METTL3) and a downstream LSM4 partnership onto SF3B4 regulation in NSCLC.","evidence":"MeRIP, co-IP for SF3B4-LSM4, rescue, xenograft","pmids":["38462740"],"confidence":"Medium","gaps":["functional meaning of SF3B4-LSM4 interaction unclear","single lab"]},{"year":2024,"claim":"Defined a direct role for SF3B4 in NMD-mediated p21 mRNA decay, explaining how its loss triggers p53-independent senescence.","evidence":"RIP for UPF1/MAGOH/RNPS1 association with p21 3'UTR, SA-β-Gal, knockdown","pmids":["39961431"],"confidence":"Medium","gaps":["mechanism of NMD-factor recruitment to p21 3'UTR not structurally resolved","single lab"]},{"year":2024,"claim":"Identified transcriptional activators of SF3B4 (SETDB1; later EP300/CREBBP), establishing how SF3B4 overexpression arises in cancer.","evidence":"Dual-luciferase reporter and ChIP for H3K27ac at the SF3B4 promoter, knockdown functional assays","pmids":["38317200","40667544"],"confidence":"Medium","gaps":["EP300/CREBBP link is Low-confidence with autophagy mediation inferred","promoter regulation contexts limited to specific cancers"]},{"year":2025,"claim":"Consolidated SF3B4 as cell-autonomously required for human neural crest survival and differentiation, with homozygous (not heterozygous) loss causing splicing-driven craniofacial defects across species.","evidence":"siRNA in hESC-derived neural crest; CRISPR sf3b4 knockout in Xenopus tropicalis with temporal RNA-seq","pmids":["40047147","40126363"],"confidence":"High","gaps":["dosage threshold reconciling haploinsufficiency disease with homozygous-only phenotype unresolved","specific causal target exons not isolated"]},{"year":2025,"claim":"Expanded the SF3B4 oncogenic splicing program to metabolism (GLS1 GAC isoform), cell cycle (CCND1 exon 2 / CDK6 holoenzyme), and migration (Talin1-FAK), each functionally validated by isoform rescue or epistasis.","evidence":"Knockdown with isoform RT-PCR, enzymatic and metabolic flux assays, co-IP for CCND1-CDK6, double-knockdown epistasis (CCND1 study is preprint)","pmids":["41411906","bio_10.1101_2025.11.25.690515","41771579"],"confidence":"Medium","gaps":["direct SF3B4 binding to each pre-mRNA not always shown","CCND1 mechanism rests on a single preprint"]},{"year":2025,"claim":"Identified SF3B4 as an essential HCC survival gene with transcriptome-wide direct targets and additional protein partners, reinforcing its splicing-centered oncogenic role.","evidence":"Genome-wide CRISPR screen in HCC organoids, iRIP-seq/long-read sequencing, co-IP (BUB1, SETD5), SREBF1 3'UTR binding by RIP/pull-down/reporter","pmids":["41071884","40234915","42255207"],"confidence":"Medium","gaps":["functional significance of BUB1 and SETD5 interactions not mechanistically dissected","many of the 252 iRIP-seq targets unvalidated"]},{"year":null,"claim":"How SF3B4's branchpoint-step splicing activity is mechanistically partitioned from its non-canonical roles in mRNA stabilization, nuclear export, and NMD-factor recruitment remains unresolved.","evidence":"No single study reconciles the spliceosomal and post-splicing activities at the structural or biochemical level","pmids":[],"confidence":"Low","gaps":["no structure of full-length SF3B4 on RNA within the spliceosome","recruitment determinants for export and NMD machinery unknown","the dosage logic linking haploinsufficiency disease to homozygous-only model phenotypes unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,2,11,13,26,31]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[1,17,22,25,29]},{"term_id":"GO:0045182","term_label":"translation regulator 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disease","url":"https://pubmed.ncbi.nlm.nih.gov/41667381","citation_count":0,"is_preprint":false},{"pmid":"41427026","id":"PMC_41427026","title":"Likely Pathogenic/Pathogenic Variants in the Spliceosome Complex Genes SNRNP200, SF3B1, SF3B2, and SF3B4 Implicated in Nonsyndromic Orofacial Cleft.","date":"2025","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/41427026","citation_count":0,"is_preprint":false},{"pmid":"41689695","id":"PMC_41689695","title":"Single-cell RNA sequencing combined with single-cell genome-wide association study identifies SF3B4 as a hub gene in hepatocellular carcinoma progression.","date":"2026","source":"Discover oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41689695","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.25.690515","title":"Transcriptome-wide profiling of alternative splicing regulators with CRISPore-seq","date":"2025-11-25","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.25.690515","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.27.630533","title":"Direct and indirect effects of spliceosome disruption compromise gene regulation by Nonsense-Mediated mRNA Decay","date":"2024-12-28","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.27.630533","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.08.25332963","title":"Modelling Acral Melanoma in Admixed Brazilians Uncovers Genomic Drivers and Targetable Pathways","date":"2025-08-12","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.08.25332963","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":28257,"output_tokens":7680,"usd":0.099986,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17164,"output_tokens":6181,"usd":0.120173,"stage2_stop_reason":"end_turn"},"total_usd":0.220159,"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\": 1997,\n      \"finding\": \"C. elegans SAP49 (SF3B4 ortholog) possesses specific RNA-binding activity residing in its second RNA recognition motif (RRM2), as demonstrated by in vitro RNA selection (SELEX).\",\n      \"method\": \"In vitro RNA selection (SELEX), RNA-binding assay\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assay with domain mapping, single lab, single organism ortholog\",\n      \"pmids\": [\"9163526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"SAP49 (SF3B4) cross-linking to retroviral pre-mRNA is enhanced at step 2 of splicing when spliceosomal progression is blocked, indicating SF3B4 regulates late-stage spliceosome transitions; UV cross-linking showed altered SAP49 binding correlates with defective transit through the last spliceosomal complex.\",\n      \"method\": \"UV cross-linking of cellular splicing factors to viral RNA substrates, spliceosomal complex analysis in cell-free splicing system\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro cell-free splicing assay with cross-linking, single lab, single study\",\n      \"pmids\": [\"9614130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The solution structure of the first RRM domain (RRM1) of human SF3B4 (SF3b49) was solved by NMR, revealing a canonical β1-α1-β2-β3-α2-β4 fold. RRM1 interacts with residues 607-616 of SF3B145 via a unique antiparallel helix-helix interaction involving SF3B4 α1; this interaction was confirmed by GST pull-down and mutational analysis.\",\n      \"method\": \"NMR structure determination, chemical shift mapping, NOESY-based docking, GST pull-down with mutational analysis\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure solved, interaction mapped by chemical shift and NOESY, validated by mutagenesis and GST pull-down in one rigorous study\",\n      \"pmids\": [\"27862552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Knockdown of Sf3b4 in Xenopus laevis embryos reduces expression of neural crest specifier genes (sox10, snail2, twist) at the neural plate border and decreases neural crest cells in pharyngeal arches, resulting in hypoplasia of neural crest-derived craniofacial cartilages. This phenotype is rescued by wild-type human SF3B4 mRNA but not by Nager syndrome mutant mRNAs, establishing a direct requirement for SF3B4 function in neural crest development.\",\n      \"method\": \"Morpholino antisense knockdown in Xenopus laevis, in situ hybridization, mRNA rescue with wild-type vs. mutant SF3B4\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — morpholino loss-of-function with defined phenotypic readout, mRNA rescue with mutant vs. wild-type discrimination, replicated across developmental stages\",\n      \"pmids\": [\"26874011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SF3B4 knockdown in liver cancer cells causes G1/S cell cycle arrest by upregulating p27Kip1 and suppressing cyclins and CDKs. SF3B4 overexpression causes aberrant splicing of the KLF4 tumor suppressor pre-mRNA, generating non-functional exon-skipped transcripts, which leads to transcriptional inactivation of p27Kip1 and activation of Slug (SNAI2).\",\n      \"method\": \"siRNA knockdown, western blotting, RT-PCR for alternative splicing, cell cycle analysis\",\n      \"journal\": \"BMB reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with defined cell cycle phenotype and splicing mechanism identified, single lab\",\n      \"pmids\": [\"29397868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"miR-133b directly targets and negatively regulates SF3B4 expression; miR-133b mimics partially abolish the effects of SF3B4 overexpression in HCC cells, and SF3B4 downstream molecules KLF4, KIP1, and SNAI2 are also modulated by miR-133b.\",\n      \"method\": \"TargetScan prediction, miRNA mimic transfection, western blotting, in vitro functional assays, xenograft mouse model\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — miRNA-target relationship confirmed by functional rescue, single lab\",\n      \"pmids\": [\"30391496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Heterozygous knockout of Sf3b4 in mice causes homeotic posteriorization of vertebral morphology, flattened calvaria, and reduced cell proliferation in the forebrain, with homozygous deletion being embryonic lethal; this establishes Sf3b4 as required for axial skeletal patterning and forebrain development in vivo.\",\n      \"method\": \"CRISPR-Cas9 knockout mouse generation, phenotypic analysis of heterozygous and homozygous mutants, histology\",\n      \"journal\": \"Developmental dynamics : an official publication of the American Association of Anatomists\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic knockout with multiple defined phenotypic readouts, homozygous lethality and heterozygous defects characterized\",\n      \"pmids\": [\"31900962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SRSF3 knockdown significantly increases SF3B4 mRNA and protein levels in HCC cells, likely by retarding SF3B4 mRNA decay rates; the coding region of SF3B4 is involved in SRSF3-mediated regulation of SF3B4 expression.\",\n      \"method\": \"siRNA knockdown, RT-qPCR, western blotting, GFP-SF3B4 fusion construct analysis\",\n      \"journal\": \"Anticancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (KD with mRNA level measurement), mechanism (mRNA stability) inferred but not directly demonstrated\",\n      \"pmids\": [\"32234894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SF3B4 knockdown in A549 NSCLC cells causes G0/G1 arrest, upregulation of p27, p21, and p53. SF3B4 interacts with UBE4B (ubiquitination factor E4B) by co-immunoprecipitation; SF3B4 depletion reduces UBE4B levels, which in turn reduces polyubiquitinated p53, leading to p53 accumulation and p21/p27 induction. Double knockdown of SF3B4 and p53 partially restores p21 expression and cell proliferation.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, western blotting, flow cytometry, double-knockdown epistasis\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reciprocal co-IP identifying SF3B4-UBE4B interaction, epistasis by double knockdown, single lab\",\n      \"pmids\": [\"35996826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-509-3p binds the 3'-UTR of SF3B4 mRNA to decrease its expression. SF3B4 promotes effective splicing of RAD52 pre-mRNA; SF3B4 loss causes intron 8 retention in RAD52 and generation of premature termination codons, reducing RAD52 protein. Decreased RAD52 partially counteracts the tumor-promoting effects of SF3B4 overexpression in ovarian cancer cells.\",\n      \"method\": \"3'-UTR luciferase reporter assay, RNA-seq/alternative splicing analysis, siRNA knockdown, in vitro and in vivo functional assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — splicing mechanism identified by RNA-seq and validated functionally by rescue experiment, single lab\",\n      \"pmids\": [\"35210412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SF3B4 promotes effective splicing of SPAG5 pre-mRNA in cervical cancer cells; SF3B4 knockdown causes intron retention and reduced maturation of SPAG5 pre-mRNA. SPAG5 deficiency impairs the oncogenic effects of SF3B4 overexpression.\",\n      \"method\": \"RNA-seq and alternative splicing analysis, siRNA knockdown, rescue experiments, in vitro and in vivo assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — splicing mechanism identified by RNA-seq and validated functionally, single lab\",\n      \"pmids\": [\"35853859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SF3B4 binds ENAH mRNA and stabilizes it (as shown by RIP and actinomycin D mRNA decay assay), leading to elevated ENAH protein which activates Notch signaling to promote HCC cell proliferation, invasion, and migration.\",\n      \"method\": \"RNA immunoprecipitation (RIP), actinomycin D mRNA stability assay, western blotting, functional cell assays\",\n      \"journal\": \"Bioengineered\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — RIP and mRNA stability assay establishing SF3B4-ENAH mRNA binding, single lab\",\n      \"pmids\": [\"35030977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SF3B4 binds GPAA1 mRNA (confirmed by RNA immunoprecipitation), and SF3B4 overexpression reverses the effects of GPAA1 knockdown on HCC cell proliferation, migration, and invasion.\",\n      \"method\": \"RNA immunoprecipitation (RIP), siRNA knockdown, rescue overexpression, functional assays\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single RIP experiment identifying RNA binding, functional link by rescue, single lab\",\n      \"pmids\": [\"35399327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SF3B4 binds the DR8 intronic cis-element of hTERT pre-mRNA (demonstrated by RNA immunoprecipitation in NSCLC cells); SF3B4 knockdown reduces full-length hTERT splicing, telomerase activity, telomere length, and cancer cell growth, establishing SF3B4 as a trans-acting splicing factor for hTERT in lung cancer.\",\n      \"method\": \"RNA immunoprecipitation (RIP), RT-PCR for hTERT isoforms, telomerase activity assay, telomere length measurement, siRNA knockdown\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — RIP demonstrating direct binding to hTERT pre-mRNA, functional consequences measured by multiple assays, single lab\",\n      \"pmids\": [\"35852380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SF3B4 promotes KLF16 mRNA export from nucleus to cytoplasm, thereby increasing KLF16 protein expression; elevated KLF16 directly binds the Twist1 promoter to activate its transcription, driving EMT and ccRCC progression.\",\n      \"method\": \"siRNA knockdown, nuclear/cytoplasmic fractionation, western blotting, chromatin immunoprecipitation (ChIP), functional migration/invasion assays, in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — fractionation demonstrating mRNA export function, ChIP for KLF16-Twist1 promoter binding, single lab\",\n      \"pmids\": [\"36639679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Heterozygous Sf3b4 knockout mice exhibit abnormal vertebral development accompanied by changes in Hox gene expression levels and patterns in somites; RNA-seq reveals widespread differential splicing events (predominantly exon skipping and intron retention) in chromatin remodeler transcripts known to regulate Hox expression, suggesting Sf3b4 controls Hox-dependent vertebral patterning via splicing of chromatin remodelers.\",\n      \"method\": \"CRISPR/Cas9 heterozygous knockout mice, RNA-seq of whole embryos and somites, in situ hybridization for Hox genes\",\n      \"journal\": \"Differentiation; research in biological diversity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo genetic model with RNA-seq identifying splicing targets, pathway placement by transcriptomic analysis, single lab\",\n      \"pmids\": [\"37167859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"sf3b4-null zebrafish display retinitis pigmentosa-like phenotypes including retinal pigment epithelium defects and rod degeneration; transcriptome analysis reveals altered retinol metabolism and retinoic acid signaling, and retinoic acid supplementation rescues key cellular phenotypes in sf3b4-/- mutants.\",\n      \"method\": \"sf3b4-/- zebrafish mutants, transcriptome profiling, immunostaining, behavioral analysis, retinoic acid rescue\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic knockout with defined retinal phenotype, pathway rescue by retinoic acid, single lab\",\n      \"pmids\": [\"37263342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Conditional homozygous deletion of Sf3b4 in neural crest cells (NCC) in mice causes craniofacial and cardiac malformations; RNA-seq prior to morphological defects reveals exon skipping in NCC regulatory network genes and in histone modifier transcripts, with enrichment of thymidine bases around branch points of affected exons, suggesting Sf3b4 regulates branchpoint-proximal splicing of NCC and cardiac developmental genes.\",\n      \"method\": \"NCC-specific conditional knockout mice, RNA-seq, splicing analysis, branchpoint sequence analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic model with tissue-specific deletion, mechanistic RNA-seq establishing splicing targets, branchpoint enrichment analysis, replicated craniofacial phenotype across models\",\n      \"pmids\": [\"39292749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SF3B4 knockdown in LUAD cells causes an alternative 5'-splice site in KAT2A pre-mRNA, reducing KAT2A at RNA and protein levels; decreased KAT2A partially reverses SF3B4-dependent LUAD growth promotion, establishing the SF3B4/KAT2A axis in lung adenocarcinoma.\",\n      \"method\": \"siRNA knockdown, RT-PCR for alternative splicing, western blotting, rescue overexpression, functional proliferation/migration assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — alternative splicing event identified and functionally validated by KAT2A rescue, single lab\",\n      \"pmids\": [\"38168564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL3 promotes SF3B4 mRNA stability through m6A modification (confirmed by MeRIP assay); SF3B4 interacts with LSM4 (confirmed by co-IP); SF3B4 depletion suppresses NSCLC cell functions that are rescued by LSM4 overexpression, placing SF3B4 upstream of LSM4 in a METTL3-SF3B4-LSM4 regulatory axis.\",\n      \"method\": \"MeRIP (m6A RIP), co-immunoprecipitation, siRNA knockdown, rescue overexpression, xenograft mouse model\",\n      \"journal\": \"Thoracic cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — MeRIP for m6A modification, Co-IP for SF3B4-LSM4 interaction, epistasis by rescue, single lab\",\n      \"pmids\": [\"38462740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SF3B4 deficiency leads to dissociation of NMD factors UPF1, MAGOH, and RNPS1 from the 3'UTR of p21 mRNA, thereby stabilizing p21 mRNA and inducing senescence via p53-independent p21 upregulation; SF3B4 recruits these NMD factors to facilitate p21 mRNA decay.\",\n      \"method\": \"siRNA knockdown, SA-β-Gal staining, RT-qPCR, western blotting, RNA immunoprecipitation for NMD factor-mRNA association\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — RIP demonstrating SF3B4-NMD factor-p21 mRNA complex, functional senescence readout, single lab\",\n      \"pmids\": [\"39961431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SETDB1 acts as a transcription factor that binds the SF3B4 promoter (confirmed by dual-luciferase reporter assay) to activate SF3B4 expression; SF3B4 knockdown impairs SETDB1-driven ovarian cancer cell proliferation and motility.\",\n      \"method\": \"Cistrome DB analysis, dual-luciferase reporter assay, siRNA knockdown, functional cell assays\",\n      \"journal\": \"Journal of ovarian research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — luciferase reporter confirming SETDB1-SF3B4 promoter interaction, epistasis by double knockdown, single lab\",\n      \"pmids\": [\"38317200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Homozygous deletion of sf3b4 in Xenopus tropicalis causes increased exon-skipping events followed by transcriptional changes affecting cranial neural crest cell migration and survival; loss of one sf3b4 copy is largely inconsequential, but homozygous loss causes major splicing defects disrupting NC-derived craniofacial cartilage.\",\n      \"method\": \"CRISPR/Cas9 sf3b4 knockout in Xenopus tropicalis, temporal RNA-seq, phenotypic characterization\",\n      \"journal\": \"Disease models & mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic model with temporal RNA-seq, replicated across Xenopus species and consistent with mouse NCC knockout\",\n      \"pmids\": [\"40126363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 siRNA knockdown in human embryonic stem cell-derived neural crest differentiation reduces neural crest gene expression, increases apoptosis, and causes premature neuronal differentiation, establishing a cell-autonomous requirement for SF3B4 in human neural crest cell survival, maintenance, and differentiation.\",\n      \"method\": \"siRNA knockdown in hESC-derived neural crest differentiation, qRT-PCR for NC markers, apoptosis assays\",\n      \"journal\": \"Developmental dynamics : an official publication of the American Association of Anatomists\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — human stem cell model with defined NC differentiation phenotype, single lab\",\n      \"pmids\": [\"40047147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 interacts with BUB1 (checkpoint protein) and regulates cancer cell mitosis and proliferation in HCC; identified by genome-wide CRISPR KO screening in patient-derived HCC organoids as a top essential survival gene.\",\n      \"method\": \"Genome-wide CRISPR knockout screen in HCC organoids, RNA immunoprecipitation-seq, long-read isoform sequencing, RNA-seq, hydrodynamic tail vein injection HCC model\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen in organoid model, multiple orthogonal transcriptomic methods, in vivo validation, single lab\",\n      \"pmids\": [\"41071884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 regulates alternative splicing of GLS1 pre-mRNA, preferentially promoting production of the high-activity GAC isoform over the KGA isoform; SF3B4 inhibition reduces GAC expression, decreases GLS enzymatic activity, impairs glutaminolysis, and suppresses glutamine-driven mitochondrial respiration in HCC cells.\",\n      \"method\": \"siRNA/shRNA knockdown, RT-PCR for GLS1 isoforms, GLS enzymatic activity assay, metabolic flux analysis, functional cell assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — isoform splicing identified and enzymatic activity measured, metabolic phenotype characterized, single lab\",\n      \"pmids\": [\"41411906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 directly binds the 3'UTR of SREBF1 mRNA (confirmed by RIP, RNA pull-down, and luciferase reporter), enhancing SREBF1 mRNA stability; SREBF1 overexpression partially rescues malignant phenotypes suppressed by SF3B4 knockdown in HCC.\",\n      \"method\": \"RNA immunoprecipitation, RNA pull-down, dual-luciferase reporter assay, mRNA stability assay, Oxford Nanopore transcriptome sequencing, xenograft model\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple orthogonal methods confirming direct 3'UTR binding and mRNA stabilization, functional rescue, single lab\",\n      \"pmids\": [\"42255207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EP300 and CREBBP activate SF3B4 expression by promoting H3K27ac modification on the SF3B4 promoter; SF3B4 knockdown-mediated inhibition of CRC cell proliferation is counteracted by enhanced autophagy.\",\n      \"method\": \"ChIP for H3K27ac at SF3B4 promoter, siRNA knockdown, autophagy assays, functional proliferation assays\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP for histone modification at SF3B4 promoter, functional link through autophagy inferred, single lab\",\n      \"pmids\": [\"40667544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 promotes melanoma cell migration through splicing-dependent regulation of Talin1; SF3B4 knockdown reduces Talin1 mRNA and protein levels, causes Talin1 intron retention, and reduces FAK phosphorylation; combined SF3B4+Talin1 knockdown shows no additive migration defect, placing them in the same pathway.\",\n      \"method\": \"siRNA knockdown, RT-qPCR, western blotting for Talin1 and phospho-FAK, wound healing and transwell migration assays, double knockdown epistasis\",\n      \"journal\": \"Cancer genomics & proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — splicing mechanism identified, epistasis by double knockdown supporting shared pathway, single lab\",\n      \"pmids\": [\"41771579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 loss triggers skipping of exon 2 in CCND1 pre-mRNA, preventing formation of a CCND1-CDK6 holoenzyme complex and blocking the G1-S transition; rescue with a CCND1 isoform containing the skipped exon restores both CDK6 complex formation and cell proliferation.\",\n      \"method\": \"CRISPore-seq (CRISPR perturbation + long-read single-cell transcriptomics), co-immunoprecipitation for CCND1-CDK6 complex, isoform rescue experiment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — isoform-level sequencing identifying specific exon skipping, co-IP for complex formation, rescue confirming mechanism, single lab preprint\",\n      \"pmids\": [\"bio_10.1101_2025.11.25.690515\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 depletion upregulates natural NMD-targeted mRNA isoforms; among spliceosome components tested, SF3B4 (along with AQR, SF3B1, and CDC40) may have a more direct role in NMD beyond indirect effects from widespread splicing disruption.\",\n      \"method\": \"Analysis of publicly available RNA-seq datasets from cells depleted of 18 spliceosome components (K562 and HepG2)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational re-analysis of existing RNA-seq datasets, no direct biochemical experiment on SF3B4's NMD role, preprint\",\n      \"pmids\": [\"bio_10.1101_2024.12.27.630533\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SF3B4 regulates HCC cell proliferation and apoptosis through alternative splicing; iRIP-seq identifies 252 direct RNA targets; SF3B4 may bind TRIM28 mRNA to modulate its expression and interact with SETD5 to influence pre-mRNA alternative splicing.\",\n      \"method\": \"iRIP-seq, RNA-seq after SF3B4 knockdown, co-immunoprecipitation\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — iRIP-seq identifying direct RNA targets with transcriptome-wide splicing analysis, single lab\",\n      \"pmids\": [\"40234915\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SF3B4 (SAP49/SF3b49) is a core subunit of the SF3b complex within the U2 snRNP whose RRM1 domain adopts a canonical fold and engages SF3B145 via a unique antiparallel helix-helix interaction; it functions at the branchpoint recognition step of pre-mRNA splicing—with loss causing predominantly exon skipping and intron retention across hundreds of transcripts—and beyond canonical splicing it stabilizes specific mRNAs via 3'UTR binding (e.g., SREBF1, ENAH, GPAA1), promotes mRNA nuclear export (KLF16), recruits NMD factors (UPF1, MAGOH, RNPS1) to target p21 mRNA for decay, and controls isoform switching of GLS1, CCND1, RAD52, SPAG5, KAT2A, and hTERT pre-mRNAs; in neural crest cells SF3B4 is cell-autonomously required for survival, migration, and differentiation, and its haploinsufficiency causes the craniofacial and cardiac defects of Nager/Rodriguez syndrome.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SF3B4 (SAP49/SF3b49) is a core RNA-binding subunit of the SF3b complex within the U2 snRNP that acts at the branchpoint recognition step of pre-mRNA splicing, where its loss produces predominantly exon skipping and intron retention across developmental and cancer transcript networks [#17, #22]. The protein contains tandem RNA recognition motifs—RRM2 carries sequence-specific RNA-binding activity, while the canonically folded RRM1 docks onto SF3B145 (SF3B3) through a unique antiparallel helix-helix interaction that anchors SF3B4 within the SF3b complex [#0, #2]. In vivo, SF3B4 governs neural crest cell survival, migration, and differentiation cell-autonomously, and its loss disrupts craniofacial cartilage formation, axial vertebral patterning through Hox-regulating chromatin remodeler splicing, and forebrain development [#3, #6, #15, #17, #22, #23]; haploinsufficiency underlies the craniofacial defects modeled by Nager-syndrome mutant mRNAs that fail to rescue SF3B4 loss [#3]. Beyond canonical splicing, SF3B4 controls isoform switching of multiple cancer-relevant pre-mRNAs—generating high-activity GLS1 GAC, full-length hTERT, exon-2-containing CCND1, and functional RAD52, SPAG5, KAT2A, and KLF4 transcripts [#4, #9, #10, #13, #18, #25, #29]—and exerts post-splicing control by stabilizing target mRNAs through 3'UTR binding (SREBF1, ENAH), promoting nuclear export of KLF16, and recruiting the NMD factors UPF1, MAGOH, and RNPS1 to drive p21 mRNA decay, thereby restraining senescence [#11, #14, #20, #26]. Through these activities SF3B4 promotes proliferation and cell-cycle progression, with depletion causing G1/S arrest via p27/p21/p53 induction, in part through interaction with UBE4B [#4, #8].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that the SF3B4 ortholog is a genuine sequence-specific RNA-binding protein and localized that activity to a defined domain, framing it as a direct RNA-engaging splicing factor rather than a passive scaffold.\",\n      \"evidence\": \"In vitro RNA selection (SELEX) on C. elegans SAP49, with domain mapping to RRM2\",\n      \"pmids\": [\"9163526\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RNA target identity in human SF3B4 not defined\", \"single ortholog, single in vitro assay\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Linked SF3B4 dynamics to spliceosomal progression, showing its binding changes at the catalytic transitions of splicing.\",\n      \"evidence\": \"UV cross-linking of SAP49 to retroviral pre-mRNA in a cell-free splicing system under spliceosomal stalling\",\n      \"pmids\": [\"9614130\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mechanism of altered binding at step 2 not resolved\", \"single substrate, single study\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved how SF3B4 is structurally integrated into the SF3b complex by solving its RRM1 fold and the molecular interface with SF3B145.\",\n      \"evidence\": \"NMR structure of human SF3B4 RRM1, chemical shift mapping, NOESY docking, GST pull-down with mutagenesis\",\n      \"pmids\": [\"27862552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"functional consequence of RRM1-SF3B145 interaction for splicing not tested in vivo\", \"RRM2 RNA-binding role not structurally addressed here\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated a direct developmental requirement for SF3B4 in neural crest specification and connected Nager-syndrome mutations to functional loss via rescue discrimination.\",\n      \"evidence\": \"Morpholino knockdown in Xenopus laevis with in situ hybridization and wild-type vs. mutant mRNA rescue\",\n      \"pmids\": [\"26874011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"splicing targets mediating the phenotype not identified\", \"morpholino specificity relies on rescue alone\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected SF3B4 to cell-cycle control and tumor biology, identifying KLF4 mis-splicing as a route to p27/Slug dysregulation.\",\n      \"evidence\": \"siRNA knockdown and overexpression in liver cancer cells, RT-PCR splicing analysis, cell cycle profiling\",\n      \"pmids\": [\"29397868\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct SF3B4 binding to KLF4 pre-mRNA not shown\", \"single cancer cell context\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified upstream control of SF3B4 by miR-133b, framing SF3B4 levels as a regulated node in HCC.\",\n      \"evidence\": \"TargetScan prediction, miRNA mimic transfection, functional and xenograft assays\",\n      \"pmids\": [\"30391496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct miRNA-SF3B4 binding inferred from prediction plus rescue\", \"single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established the in vivo requirement of Sf3b4 for axial skeletal patterning and forebrain development through a mammalian genetic model, with homozygous loss being lethal.\",\n      \"evidence\": \"CRISPR-Cas9 Sf3b4 knockout mice, phenotypic and histological analysis\",\n      \"pmids\": [\"31900962\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"molecular splicing targets not defined in this study\", \"dosage-sensitivity mechanism unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Added a layer of expression control by linking SRSF3 to SF3B4 mRNA stability.\",\n      \"evidence\": \"siRNA knockdown, RT-qPCR, western blotting, GFP-SF3B4 construct analysis in HCC\",\n      \"pmids\": [\"32234894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mRNA decay mechanism inferred not directly measured\", \"single method, single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a protein-level mechanism by which SF3B4 restrains p53, showing SF3B4-UBE4B interaction sustains p53 polyubiquitination and thereby proliferation.\",\n      \"evidence\": \"Reciprocal co-IP, double-knockdown epistasis, flow cytometry in A549 NSCLC cells\",\n      \"pmids\": [\"35996826\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"whether interaction is direct or splicing-independent unclear\", \"single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Built a recurring paradigm of SF3B4-dependent productive splicing of oncogenic targets, here for RAD52 and SPAG5, where loss causes intron retention and PTC generation.\",\n      \"evidence\": \"3'UTR luciferase, RNA-seq splicing analysis, knockdown and functional rescue in ovarian and cervical cancer cells\",\n      \"pmids\": [\"35210412\", \"35853859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct SF3B4 binding to these pre-mRNAs not always shown\", \"single lab per target\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended SF3B4 function beyond splicing into mRNA stabilization via 3'UTR binding, establishing a post-transcriptional regulatory mode (ENAH, GPAA1).\",\n      \"evidence\": \"RNA immunoprecipitation, actinomycin D decay assays, rescue functional assays in HCC\",\n      \"pmids\": [\"35030977\", \"35399327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"how a spliceosomal factor stabilizes cytoplasmic mRNA mechanistically unclear\", \"GPAA1 link rests on single RIP plus rescue\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed SF3B4 acts as a trans-acting splicing factor for hTERT via a defined intronic cis-element, coupling it to telomerase activity.\",\n      \"evidence\": \"RIP to the DR8 element, hTERT isoform RT-PCR, telomerase and telomere assays in NSCLC\",\n      \"pmids\": [\"35852380\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"structural basis of DR8 recognition unknown\", \"single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed an mRNA nuclear-export function for SF3B4, driving KLF16-Twist1-mediated EMT.\",\n      \"evidence\": \"Nuclear/cytoplasmic fractionation, ChIP for KLF16-Twist1, xenograft in ccRCC\",\n      \"pmids\": [\"36639679\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"export machinery SF3B4 engages not identified\", \"directness of KLF16 mRNA binding not shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected the in vivo skeletal phenotype to a splicing mechanism, implicating mis-splicing of chromatin remodelers that regulate Hox genes.\",\n      \"evidence\": \"Heterozygous Sf3b4 knockout mice, RNA-seq of somites, Hox in situ hybridization\",\n      \"pmids\": [\"37167859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"causal chromatin-remodeler target not pinpointed\", \"correlative transcriptomic placement\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Broadened the in vivo phenotypic spectrum to retinal degeneration with a metabolic-pathway link, rescued by retinoic acid.\",\n      \"evidence\": \"sf3b4-null zebrafish, transcriptomics, retinoic acid rescue\",\n      \"pmids\": [\"37263342\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"splicing targets driving retinol metabolism changes not defined\", \"single model\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the strongest mechanistic placement of SF3B4 at branchpoint-proximal splicing in vivo, tying neural crest and cardiac developmental gene networks to exon skipping.\",\n      \"evidence\": \"NCC-specific conditional knockout mice, RNA-seq, branchpoint sequence enrichment analysis\",\n      \"pmids\": [\"39292749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"individual causal target exons among NCC genes not isolated\", \"thymidine/branchpoint enrichment correlative\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Added KAT2A isoform control to the SF3B4 splicing-target repertoire in lung adenocarcinoma.\",\n      \"evidence\": \"siRNA knockdown, alternative 5'-splice-site RT-PCR, functional rescue\",\n      \"pmids\": [\"38168564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct pre-mRNA binding not demonstrated\", \"single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Layered m6A-dependent stabilization (METTL3) and a downstream LSM4 partnership onto SF3B4 regulation in NSCLC.\",\n      \"evidence\": \"MeRIP, co-IP for SF3B4-LSM4, rescue, xenograft\",\n      \"pmids\": [\"38462740\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"functional meaning of SF3B4-LSM4 interaction unclear\", \"single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a direct role for SF3B4 in NMD-mediated p21 mRNA decay, explaining how its loss triggers p53-independent senescence.\",\n      \"evidence\": \"RIP for UPF1/MAGOH/RNPS1 association with p21 3'UTR, SA-β-Gal, knockdown\",\n      \"pmids\": [\"39961431\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mechanism of NMD-factor recruitment to p21 3'UTR not structurally resolved\", \"single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified transcriptional activators of SF3B4 (SETDB1; later EP300/CREBBP), establishing how SF3B4 overexpression arises in cancer.\",\n      \"evidence\": \"Dual-luciferase reporter and ChIP for H3K27ac at the SF3B4 promoter, knockdown functional assays\",\n      \"pmids\": [\"38317200\", \"40667544\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"EP300/CREBBP link is Low-confidence with autophagy mediation inferred\", \"promoter regulation contexts limited to specific cancers\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Consolidated SF3B4 as cell-autonomously required for human neural crest survival and differentiation, with homozygous (not heterozygous) loss causing splicing-driven craniofacial defects across species.\",\n      \"evidence\": \"siRNA in hESC-derived neural crest; CRISPR sf3b4 knockout in Xenopus tropicalis with temporal RNA-seq\",\n      \"pmids\": [\"40047147\", \"40126363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"dosage threshold reconciling haploinsufficiency disease with homozygous-only phenotype unresolved\", \"specific causal target exons not isolated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded the SF3B4 oncogenic splicing program to metabolism (GLS1 GAC isoform), cell cycle (CCND1 exon 2 / CDK6 holoenzyme), and migration (Talin1-FAK), each functionally validated by isoform rescue or epistasis.\",\n      \"evidence\": \"Knockdown with isoform RT-PCR, enzymatic and metabolic flux assays, co-IP for CCND1-CDK6, double-knockdown epistasis (CCND1 study is preprint)\",\n      \"pmids\": [\"41411906\", \"bio_10.1101_2025.11.25.690515\", \"41771579\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct SF3B4 binding to each pre-mRNA not always shown\", \"CCND1 mechanism rests on a single preprint\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified SF3B4 as an essential HCC survival gene with transcriptome-wide direct targets and additional protein partners, reinforcing its splicing-centered oncogenic role.\",\n      \"evidence\": \"Genome-wide CRISPR screen in HCC organoids, iRIP-seq/long-read sequencing, co-IP (BUB1, SETD5), SREBF1 3'UTR binding by RIP/pull-down/reporter\",\n      \"pmids\": [\"41071884\", \"40234915\", \"42255207\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"functional significance of BUB1 and SETD5 interactions not mechanistically dissected\", \"many of the 252 iRIP-seq targets unvalidated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SF3B4's branchpoint-step splicing activity is mechanistically partitioned from its non-canonical roles in mRNA stabilization, nuclear export, and NMD-factor recruitment remains unresolved.\",\n      \"evidence\": \"No single study reconciles the spliceosomal and post-splicing activities at the structural or biochemical level\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"no structure of full-length SF3B4 on RNA within the spliceosome\", \"recruitment determinants for export and NMD machinery unknown\", \"the dosage logic linking haploinsufficiency disease to homozygous-only model phenotypes unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 2, 11, 13, 26, 31]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [1, 17, 22, 25, 29]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [11, 20, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [14, 17]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [2, 17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [1, 2, 17, 22]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 6, 17, 22, 23]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 8, 29]}\n    ],\n    \"complexes\": [\"SF3b complex\", \"U2 snRNP\"],\n    \"partners\": [\"SF3B3\", \"UBE4B\", \"LSM4\", \"UPF1\", \"MAGOH\", \"RNPS1\", \"BUB1\", \"SETD5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":5,"faith_pct":80.0}}