{"gene":"WDR33","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2014,"finding":"WDR33 directly contacts the AAUAAA polyadenylation signal RNA in vitro and in vivo, and is required for CPSF binding to AAUAAA-containing RNA; WDR33 can be specifically UV cross-linked to AAUAAA-containing RNAs, and PAR-CLIP showed WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity.","method":"In vitro reconstitution of CPSF subcomplex, UV cross-linking, PAR-CLIP, polyadenylation assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro with purified proteins, UV cross-linking, and transcriptome-wide PAR-CLIP; replicated in companion paper (PMID:25301780)","pmids":["25301781"],"is_preprint":false},{"year":2014,"finding":"CPSF30 (CPSF4) and WDR33 directly contact the AAUAAA polyadenylation signal; the CPSF30-RNA interaction is primarily mediated by zinc fingers 2 and 3 and is essential for mRNA 3' processing; the influenza NS1A protein targets these zinc fingers to suppress host mRNA 3' processing.","method":"In vitro and in vivo UV cross-linking, mutagenesis of zinc finger domains, iCLIP, mRNA 3' processing assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro assays with mutagenesis and in vivo cross-linking; replicated in companion paper (PMID:25301781)","pmids":["25301780"],"is_preprint":false},{"year":2014,"finding":"Only four subunits of CPSF—CPSF160, CPSF30, hFip1, and WDR33—are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation; CPSF100, CPSF73, and symplekin are dispensable for this minimal activity.","method":"Reconstitution of recombinant CPSF subcomplex with purified proteins, AAUAAA-dependent polyadenylation assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution with defined subunit composition, replicated across two labs","pmids":["25301781"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structure at 3.4 Å of the quaternary complex CPSF-160–WDR33–CPSF-30–AAUAAA RNA revealed that U3 and A6 bases of AAUAAA form an intramolecular Hoogsteen base pair and directly contact WDR33, while A1/A2 are recognized by ZF2 and A4/A5 by ZF3 of CPSF-30; CPSF-160 functions as a scaffold to preorganize CPSF-30 and WDR33 for high-affinity AAUAAA binding.","method":"Cryo-electron microscopy structure determination at 3.4 Å resolution","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with atomic-level detail of protein-RNA contacts, replicated by Clerici et al. 2017","pmids":["29208711"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of the CPSF160–WDR33 subcomplex and cross-linking/mass spectrometry defined molecular architecture of the core CPSF complex; an N-terminal lysine/arginine-rich (KR-rich) motif in WDR33 was identified as a critical determinant of specific AAUAAA motif recognition by quantitative RNA-binding assays.","method":"Crystal structure determination, cross-linking coupled mass spectrometry (XL-MS), fluorescence anisotropy RNA-binding assays, mutagenesis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus orthogonal biochemical methods (XL-MS and fluorescence anisotropy with mutagenesis) in single study","pmids":["29274231"],"is_preprint":false},{"year":2019,"finding":"The CPSF-160–WDR33–CPSF-30 ternary complex binds AAUAAA with ~3 nM affinity; CPSF-30 and WDR33 are both required for high-affinity PAS binding, as their binary complexes with CPSF-160 alone have much lower affinity; mutations of CPSF-30 residues with van der Waals contacts to AAUAAA bases substantially reduce affinity.","method":"Fluorescence polarization binding assays with purified recombinant proteins, systematic sequence and mutagenesis analysis","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative in vitro binding assays with systematic mutagenesis in a single rigorous study","pmids":["31462423"],"is_preprint":false},{"year":2022,"finding":"RBBP6 activates human CPSF endonuclease activity and interacts with the WDR33 and CPSF73 subunits of CPSF; unlike its yeast homolog Mpe1, RBBP6 does not co-purify stably with CPSF but is recruited in an RNA-dependent manner.","method":"Biochemical reconstitution of endonuclease activity with purified proteins, sequence and mutational analysis of RBBP6-WDR33 and RBBP6-CPSF73 interactions","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reconstituted cleavage activity and mutational analysis in one study, interaction with WDR33 inferred from mutational/sequence analysis rather than direct structural data","pmids":["35177536"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structure of human mPSF (CPSF160–WDR33–CPSF30–Fip1) with AUUAAA PAS revealed conformational differences in A1 and U2 nucleotides compared to AAUAAA, with U2 base making two hydrogen bonds with ZF2 of CPSF30, while the WDR33 and remaining nucleotide contact modes are essentially identical to those for AAUAAA.","method":"Cryo-electron microscopy structure determination","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution cryo-EM structure with detailed atomic contacts, single lab but rigorous structural method","pmids":["36130077"],"is_preprint":false},{"year":2024,"finding":"Non-canonical WDR33 isoforms V2 and V3 (generated by alternative polyadenylation within promoter-proximal introns) localize to the endoplasmic reticulum and interact with STING; V2 suppresses interferon-β induction by preventing STING disulfide oligomerization and promotes autophagy by recruiting WIPI2 isoforms; V3 increases STING protein levels.","method":"Subcellular fractionation/localization, co-immunoprecipitation, isoform-specific knockdown/overexpression, interferon-β reporter assays, autophagy assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal assays (Co-IP, localization, functional reporter) in single lab study","pmids":["38430516"],"is_preprint":false},{"year":2024,"finding":"WDR33 alternative polyadenylation producing V2 and V3 isoforms is regulated by CFIm25 levels; V2 production is enabled by inefficient splicing of intron 6 allowing weak PA sites to be used; V3 PA site usage is limited by highly efficient splicing of intron 7 and dependency on an alternative 3' splice site.","method":"CFIm25 knockdown/overexpression, newly developed splicing and PA site strength assays, isoform quantification","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional knockdown with multiple mechanistic assays in single lab study","pmids":["39327832"],"is_preprint":false},{"year":2001,"finding":"WDR33 (WDC146) protein is localized to the nucleus and its mRNA is most highly expressed in testis, specifically in pachytene spermatocytes as shown by in situ hybridization.","method":"Northern blot, in situ hybridization, subcellular localization by immunostaining","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment with tissue-specific expression; foundational characterization paper","pmids":["11162572"],"is_preprint":false},{"year":2019,"finding":"WDR33 and CPSF4 polyadenylation factors translocate to the nucleus upon macrophage stimulation, and knockdown of WDR33 prevents nuclear localization of NFκB in stimulated macrophages, indicating WDR33 is required for the inflammatory response in macrophages.","method":"siRNA knockdown, immunofluorescence localization, NFκB nuclear translocation assay in macrophages","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (knockdown + localization), indirect functional link to NFκB pathway","pmids":["30886197"],"is_preprint":false},{"year":2024,"finding":"SNORD51 can competitively bind to WDR33 with the 3'UTR of ZBED6 pre-mRNA, thereby inhibiting 3'-end processing of ZBED6 pre-mRNA and reducing ZBED6 mRNA expression in glioblastoma cells.","method":"RNA immunoprecipitation, competitive binding assay, knockdown experiments, gene expression analysis","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, mechanistic detail limited in abstract, competition assay not fully characterized","pmids":["39516455"],"is_preprint":false},{"year":2024,"finding":"WDR33 and CPSF30 (mPSF subunits) contribute to polyadenylation of SINE transcripts generated by RNA polymerase III in an AAUAAA-dependent manner, while CPSF100, CPSF73, and symplekin (mCF subcomplex) are not required for this activity.","method":"siRNA knockdown of individual CPSF components in HeLa cells, Northern hybridization of SINE transcript polyadenylation","journal":"Molekuliarnaia biologiia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic knockdown of multiple subunits with direct readout, single lab study","pmids":["39707854"],"is_preprint":false}],"current_model":"WDR33 is a core subunit of the mammalian polyadenylation specificity factor (mPSF) that directly recognizes the AAUAAA polyadenylation signal through its N-terminal KR-rich motif (specifically contacting U3 and A6 via a Hoogsteen base pair), acts cooperatively with CPSF30 zinc fingers within a CPSF160-scaffolded quaternary complex to achieve ~3 nM affinity for AAUAAA, and is necessary for pre-mRNA 3'-end cleavage and polyadenylation; additionally, non-canonical alternatively polyadenylated WDR33 isoforms (V2/V3) localize to the ER and regulate STING-mediated innate immune responses independently of polyadenylation."},"narrative":{"mechanistic_narrative":"WDR33 is a core subunit of the mammalian polyadenylation specificity factor (mPSF) that directly recognizes the AAUAAA polyadenylation signal and is essential for pre-mRNA 3'-end cleavage and polyadenylation [PMID:25301781]. Within a minimal CPSF subcomplex consisting of CPSF160, CPSF30, hFip1, and WDR33—sufficient to reconstitute AAUAAA-dependent polyadenylation—WDR33 makes direct, sequence-specific contacts with the signal RNA both in vitro and in vivo [PMID:25301781]. Structural work showed that CPSF160 acts as a scaffold preorganizing WDR33 and CPSF30 around the RNA, with the U3 and A6 bases of AAUAAA forming an intramolecular Hoogsteen base pair that is read out directly by WDR33, while CPSF30 zinc fingers 2 and 3 recognize the flanking adenosines [PMID:29208711, PMID:25301780]; an N-terminal lysine/arginine-rich (KR-rich) motif in WDR33 is the critical determinant of specific signal recognition [PMID:29274231]. WDR33 and CPSF30 act cooperatively, and only the ternary CPSF160–WDR33–CPSF30 complex achieves high (~3 nM) affinity for AAUAAA, with binary complexes binding far more weakly [PMID:31462423]. WDR33 also interacts with RBBP6, which is recruited in an RNA-dependent manner to activate CPSF endonuclease cleavage [PMID:35177536], and the same machinery polyadenylates Pol III-derived SINE transcripts in an AAUAAA-dependent fashion [PMID:39707854]. Distinct from its canonical role, non-canonical alternatively polyadenylated WDR33 isoforms (V2/V3) generated within promoter-proximal introns localize to the endoplasmic reticulum and modulate STING-dependent innate immune signaling and autophagy, with isoform choice governed by CFIm25 and intronic splicing efficiency [PMID:38430516, PMID:39327832].","teleology":[{"year":2014,"claim":"Established that WDR33 is not merely a CPSF accessory subunit but a direct sequence-specific reader of the AAUAAA polyadenylation signal, answering how CPSF recognizes the canonical PAS.","evidence":"in vitro reconstitution of a CPSF subcomplex, UV cross-linking, and transcriptome-wide PAR-CLIP","pmids":["25301781"],"confidence":"High","gaps":["Did not resolve the atomic contacts between WDR33 and individual signal bases","Did not define which WDR33 region mediates RNA contact"]},{"year":2014,"claim":"Defined the cooperative RNA-contacting partners of WDR33 by showing CPSF30 zinc fingers 2 and 3 also contact AAUAAA and are essential for 3' processing, also explaining how influenza NS1A subverts host polyadenylation.","evidence":"in vitro/in vivo UV cross-linking, zinc-finger mutagenesis, iCLIP, and 3' processing assays","pmids":["25301780"],"confidence":"High","gaps":["Stoichiometry and geometry of joint WDR33/CPSF30 RNA engagement unresolved","Did not establish affinity contributions of each subunit"]},{"year":2014,"claim":"Reduced the AAUAAA-recognition machinery to a minimal functional module by showing only CPSF160, CPSF30, hFip1 and WDR33 are necessary and sufficient for AAUAAA-dependent polyadenylation, separating signal recognition from the cleavage subunits.","evidence":"reconstitution of recombinant CPSF subcomplex and AAUAAA-dependent polyadenylation assay","pmids":["25301781"],"confidence":"High","gaps":["Did not address how this subcomplex couples to the catalytic cleavage module in vivo"]},{"year":2017,"claim":"Provided the structural mechanism of PAS recognition, showing CPSF160 scaffolds WDR33 and CPSF30 and that U3/A6 form a Hoogsteen base pair read by WDR33, and identified the WDR33 KR-rich motif as the specificity determinant.","evidence":"3.4 Å cryo-EM of CPSF160–WDR33–CPSF30–AAUAAA, plus crystal structure, XL-MS and fluorescence anisotropy binding assays","pmids":["29208711","29274231"],"confidence":"High","gaps":["Did not quantify the absolute affinity contribution of each subunit","Did not capture the assembled cleavage-competent holo-complex"]},{"year":2019,"claim":"Quantified the cooperativity established structurally, showing the ternary CPSF160–WDR33–CPSF30 complex binds AAUAAA at ~3 nM whereas binary complexes are much weaker, demonstrating that both WDR33 and CPSF30 are required for high-affinity recognition.","evidence":"fluorescence polarization binding assays with purified proteins and systematic mutagenesis","pmids":["31462423"],"confidence":"High","gaps":["Did not address kinetics or signal-variant discrimination in vivo"]},{"year":2022,"claim":"Connected the WDR33 recognition module to catalysis by showing RBBP6 binds WDR33 and CPSF73 and activates CPSF endonuclease activity through RNA-dependent recruitment.","evidence":"biochemical reconstitution of endonuclease activity and mutational analysis of RBBP6 interactions","pmids":["35177536"],"confidence":"Medium","gaps":["WDR33–RBBP6 interaction inferred from mutational/sequence analysis rather than direct structure","Structural basis of endonuclease activation not resolved"]},{"year":2022,"claim":"Explained how WDR33-containing mPSF accommodates the variant AUUAAA signal, showing CPSF30 ZF2 adapts via U2 hydrogen bonds while WDR33 contacts remain unchanged.","evidence":"cryo-EM of human mPSF (CPSF160–WDR33–CPSF30–Fip1) with AUUAAA","pmids":["36130077"],"confidence":"High","gaps":["Did not survey the full spectrum of non-canonical signals","Functional consequences for variant-signal genes not assessed"]},{"year":2024,"claim":"Extended WDR33 substrate scope beyond Pol II mRNAs by showing it and CPSF30 polyadenylate Pol III-derived SINE transcripts in an AAUAAA-dependent manner without the cleavage subcomplex.","evidence":"siRNA knockdown of CPSF components and Northern hybridization in HeLa cells","pmids":["39707854"],"confidence":"Medium","gaps":["Mechanism of mPSF recruitment to Pol III transcripts unclear","Biological role of SINE polyadenylation not defined"]},{"year":2024,"claim":"Revealed a polyadenylation-independent function by showing alternatively polyadenylated WDR33 isoforms V2/V3 localize to the ER, bind STING, and modulate interferon-β induction and autophagy, with isoform production controlled by CFIm25 and intronic splicing.","evidence":"subcellular fractionation, Co-IP, isoform-specific knockdown/overexpression, IFN-β reporter and autophagy assays, plus CFIm25 manipulation and splicing/PA-strength assays","pmids":["38430516","39327832"],"confidence":"Medium","gaps":["Single-lab functional study without independent replication","Direct structural basis of WDR33–STING interaction unknown","Physiological relevance in vivo not established"]},{"year":null,"claim":"How WDR33's canonical 3'-processing role is integrated with its non-canonical ER/STING isoform functions, and what governs its testis- and stimulation-dependent expression in physiology, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No in vivo genetic model linking WDR33 to organismal phenotype in the corpus","Relationship between nuclear polyadenylation and immune isoform functions uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,1,3,4,5,7]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[2,6,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[10,11]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,2,3,5]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,2]}],"complexes":["mPSF (CPSF160–WDR33–CPSF30–Fip1)","CPSF"],"partners":["CPSF160","CPSF30","FIP1L1","RBBP6","CPSF73","STING1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9C0J8","full_name":"pre-mRNA 3' end processing protein WDR33","aliases":["WD repeat-containing protein 33","WD repeat-containing protein of 146 kDa"],"length_aa":1336,"mass_kda":145.9,"function":"Essential for both cleavage and polyadenylation of pre-mRNA 3' ends","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9C0J8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/WDR33","classification":"Common Essential","n_dependent_lines":1201,"n_total_lines":1208,"dependency_fraction":0.9942052980132451},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"RBM14","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"TOP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/WDR33","total_profiled":1310},"omim":[{"mim_id":"618082","title":"WD REPEAT-CONTAINING PROTEIN 33; WDR33","url":"https://www.omim.org/entry/618082"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Nucleoli fibrillar center","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/WDR33"},"hgnc":{"alias_symbol":["FLJ11294","WDC146","NET14"],"prev_symbol":[]},"alphafold":{"accession":"Q9C0J8","domains":[{"cath_id":"2.130.10.10","chopping":"102-404","consensus_level":"medium","plddt":96.6272,"start":102,"end":404}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0J8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0J8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0J8-F1-predicted_aligned_error_v6.png","plddt_mean":55.41},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=WDR33","jax_strain_url":"https://www.jax.org/strain/search?query=WDR33"},"sequence":{"accession":"Q9C0J8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9C0J8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9C0J8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0J8"}},"corpus_meta":[{"pmid":"25301780","id":"PMC_25301780","title":"CPSF30 and 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brucellosis cases.","date":"2024","source":"Diagnostic microbiology and infectious disease","url":"https://pubmed.ncbi.nlm.nih.gov/39550977","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14584,"output_tokens":3478,"usd":0.047961,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10907,"output_tokens":3474,"usd":0.070693,"stage2_stop_reason":"end_turn"},"total_usd":0.118654,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"WDR33 directly contacts the AAUAAA polyadenylation signal RNA in vitro and in vivo, and is required for CPSF binding to AAUAAA-containing RNA; WDR33 can be specifically UV cross-linked to AAUAAA-containing RNAs, and PAR-CLIP showed WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity.\",\n      \"method\": \"In vitro reconstitution of CPSF subcomplex, UV cross-linking, PAR-CLIP, polyadenylation assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro with purified proteins, UV cross-linking, and transcriptome-wide PAR-CLIP; replicated in companion paper (PMID:25301780)\",\n      \"pmids\": [\"25301781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CPSF30 (CPSF4) and WDR33 directly contact the AAUAAA polyadenylation signal; the CPSF30-RNA interaction is primarily mediated by zinc fingers 2 and 3 and is essential for mRNA 3' processing; the influenza NS1A protein targets these zinc fingers to suppress host mRNA 3' processing.\",\n      \"method\": \"In vitro and in vivo UV cross-linking, mutagenesis of zinc finger domains, iCLIP, mRNA 3' processing assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro assays with mutagenesis and in vivo cross-linking; replicated in companion paper (PMID:25301781)\",\n      \"pmids\": [\"25301780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Only four subunits of CPSF—CPSF160, CPSF30, hFip1, and WDR33—are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation; CPSF100, CPSF73, and symplekin are dispensable for this minimal activity.\",\n      \"method\": \"Reconstitution of recombinant CPSF subcomplex with purified proteins, AAUAAA-dependent polyadenylation assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution with defined subunit composition, replicated across two labs\",\n      \"pmids\": [\"25301781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structure at 3.4 Å of the quaternary complex CPSF-160–WDR33–CPSF-30–AAUAAA RNA revealed that U3 and A6 bases of AAUAAA form an intramolecular Hoogsteen base pair and directly contact WDR33, while A1/A2 are recognized by ZF2 and A4/A5 by ZF3 of CPSF-30; CPSF-160 functions as a scaffold to preorganize CPSF-30 and WDR33 for high-affinity AAUAAA binding.\",\n      \"method\": \"Cryo-electron microscopy structure determination at 3.4 Å resolution\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with atomic-level detail of protein-RNA contacts, replicated by Clerici et al. 2017\",\n      \"pmids\": [\"29208711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of the CPSF160–WDR33 subcomplex and cross-linking/mass spectrometry defined molecular architecture of the core CPSF complex; an N-terminal lysine/arginine-rich (KR-rich) motif in WDR33 was identified as a critical determinant of specific AAUAAA motif recognition by quantitative RNA-binding assays.\",\n      \"method\": \"Crystal structure determination, cross-linking coupled mass spectrometry (XL-MS), fluorescence anisotropy RNA-binding assays, mutagenesis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus orthogonal biochemical methods (XL-MS and fluorescence anisotropy with mutagenesis) in single study\",\n      \"pmids\": [\"29274231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The CPSF-160–WDR33–CPSF-30 ternary complex binds AAUAAA with ~3 nM affinity; CPSF-30 and WDR33 are both required for high-affinity PAS binding, as their binary complexes with CPSF-160 alone have much lower affinity; mutations of CPSF-30 residues with van der Waals contacts to AAUAAA bases substantially reduce affinity.\",\n      \"method\": \"Fluorescence polarization binding assays with purified recombinant proteins, systematic sequence and mutagenesis analysis\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative in vitro binding assays with systematic mutagenesis in a single rigorous study\",\n      \"pmids\": [\"31462423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBBP6 activates human CPSF endonuclease activity and interacts with the WDR33 and CPSF73 subunits of CPSF; unlike its yeast homolog Mpe1, RBBP6 does not co-purify stably with CPSF but is recruited in an RNA-dependent manner.\",\n      \"method\": \"Biochemical reconstitution of endonuclease activity with purified proteins, sequence and mutational analysis of RBBP6-WDR33 and RBBP6-CPSF73 interactions\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reconstituted cleavage activity and mutational analysis in one study, interaction with WDR33 inferred from mutational/sequence analysis rather than direct structural data\",\n      \"pmids\": [\"35177536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structure of human mPSF (CPSF160–WDR33–CPSF30–Fip1) with AUUAAA PAS revealed conformational differences in A1 and U2 nucleotides compared to AAUAAA, with U2 base making two hydrogen bonds with ZF2 of CPSF30, while the WDR33 and remaining nucleotide contact modes are essentially identical to those for AAUAAA.\",\n      \"method\": \"Cryo-electron microscopy structure determination\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution cryo-EM structure with detailed atomic contacts, single lab but rigorous structural method\",\n      \"pmids\": [\"36130077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Non-canonical WDR33 isoforms V2 and V3 (generated by alternative polyadenylation within promoter-proximal introns) localize to the endoplasmic reticulum and interact with STING; V2 suppresses interferon-β induction by preventing STING disulfide oligomerization and promotes autophagy by recruiting WIPI2 isoforms; V3 increases STING protein levels.\",\n      \"method\": \"Subcellular fractionation/localization, co-immunoprecipitation, isoform-specific knockdown/overexpression, interferon-β reporter assays, autophagy assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal assays (Co-IP, localization, functional reporter) in single lab study\",\n      \"pmids\": [\"38430516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"WDR33 alternative polyadenylation producing V2 and V3 isoforms is regulated by CFIm25 levels; V2 production is enabled by inefficient splicing of intron 6 allowing weak PA sites to be used; V3 PA site usage is limited by highly efficient splicing of intron 7 and dependency on an alternative 3' splice site.\",\n      \"method\": \"CFIm25 knockdown/overexpression, newly developed splicing and PA site strength assays, isoform quantification\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional knockdown with multiple mechanistic assays in single lab study\",\n      \"pmids\": [\"39327832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"WDR33 (WDC146) protein is localized to the nucleus and its mRNA is most highly expressed in testis, specifically in pachytene spermatocytes as shown by in situ hybridization.\",\n      \"method\": \"Northern blot, in situ hybridization, subcellular localization by immunostaining\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment with tissue-specific expression; foundational characterization paper\",\n      \"pmids\": [\"11162572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WDR33 and CPSF4 polyadenylation factors translocate to the nucleus upon macrophage stimulation, and knockdown of WDR33 prevents nuclear localization of NFκB in stimulated macrophages, indicating WDR33 is required for the inflammatory response in macrophages.\",\n      \"method\": \"siRNA knockdown, immunofluorescence localization, NFκB nuclear translocation assay in macrophages\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (knockdown + localization), indirect functional link to NFκB pathway\",\n      \"pmids\": [\"30886197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SNORD51 can competitively bind to WDR33 with the 3'UTR of ZBED6 pre-mRNA, thereby inhibiting 3'-end processing of ZBED6 pre-mRNA and reducing ZBED6 mRNA expression in glioblastoma cells.\",\n      \"method\": \"RNA immunoprecipitation, competitive binding assay, knockdown experiments, gene expression analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanistic detail limited in abstract, competition assay not fully characterized\",\n      \"pmids\": [\"39516455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"WDR33 and CPSF30 (mPSF subunits) contribute to polyadenylation of SINE transcripts generated by RNA polymerase III in an AAUAAA-dependent manner, while CPSF100, CPSF73, and symplekin (mCF subcomplex) are not required for this activity.\",\n      \"method\": \"siRNA knockdown of individual CPSF components in HeLa cells, Northern hybridization of SINE transcript polyadenylation\",\n      \"journal\": \"Molekuliarnaia biologiia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic knockdown of multiple subunits with direct readout, single lab study\",\n      \"pmids\": [\"39707854\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"WDR33 is a core subunit of the mammalian polyadenylation specificity factor (mPSF) that directly recognizes the AAUAAA polyadenylation signal through its N-terminal KR-rich motif (specifically contacting U3 and A6 via a Hoogsteen base pair), acts cooperatively with CPSF30 zinc fingers within a CPSF160-scaffolded quaternary complex to achieve ~3 nM affinity for AAUAAA, and is necessary for pre-mRNA 3'-end cleavage and polyadenylation; additionally, non-canonical alternatively polyadenylated WDR33 isoforms (V2/V3) localize to the ER and regulate STING-mediated innate immune responses independently of polyadenylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"WDR33 is a core subunit of the mammalian polyadenylation specificity factor (mPSF) that directly recognizes the AAUAAA polyadenylation signal and is essential for pre-mRNA 3'-end cleavage and polyadenylation [#0, #2]. Within a minimal CPSF subcomplex consisting of CPSF160, CPSF30, hFip1, and WDR33—sufficient to reconstitute AAUAAA-dependent polyadenylation—WDR33 makes direct, sequence-specific contacts with the signal RNA both in vitro and in vivo [#0, #2]. Structural work showed that CPSF160 acts as a scaffold preorganizing WDR33 and CPSF30 around the RNA, with the U3 and A6 bases of AAUAAA forming an intramolecular Hoogsteen base pair that is read out directly by WDR33, while CPSF30 zinc fingers 2 and 3 recognize the flanking adenosines [#3, #1]; an N-terminal lysine/arginine-rich (KR-rich) motif in WDR33 is the critical determinant of specific signal recognition [#4]. WDR33 and CPSF30 act cooperatively, and only the ternary CPSF160–WDR33–CPSF30 complex achieves high (~3 nM) affinity for AAUAAA, with binary complexes binding far more weakly [#5]. WDR33 also interacts with RBBP6, which is recruited in an RNA-dependent manner to activate CPSF endonuclease cleavage [#6], and the same machinery polyadenylates Pol III-derived SINE transcripts in an AAUAAA-dependent fashion [#13]. Distinct from its canonical role, non-canonical alternatively polyadenylated WDR33 isoforms (V2/V3) generated within promoter-proximal introns localize to the endoplasmic reticulum and modulate STING-dependent innate immune signaling and autophagy, with isoform choice governed by CFIm25 and intronic splicing efficiency [#8, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Established that WDR33 is not merely a CPSF accessory subunit but a direct sequence-specific reader of the AAUAAA polyadenylation signal, answering how CPSF recognizes the canonical PAS.\",\n      \"evidence\": \"in vitro reconstitution of a CPSF subcomplex, UV cross-linking, and transcriptome-wide PAR-CLIP\",\n      \"pmids\": [\"25301781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the atomic contacts between WDR33 and individual signal bases\", \"Did not define which WDR33 region mediates RNA contact\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the cooperative RNA-contacting partners of WDR33 by showing CPSF30 zinc fingers 2 and 3 also contact AAUAAA and are essential for 3' processing, also explaining how influenza NS1A subverts host polyadenylation.\",\n      \"evidence\": \"in vitro/in vivo UV cross-linking, zinc-finger mutagenesis, iCLIP, and 3' processing assays\",\n      \"pmids\": [\"25301780\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and geometry of joint WDR33/CPSF30 RNA engagement unresolved\", \"Did not establish affinity contributions of each subunit\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Reduced the AAUAAA-recognition machinery to a minimal functional module by showing only CPSF160, CPSF30, hFip1 and WDR33 are necessary and sufficient for AAUAAA-dependent polyadenylation, separating signal recognition from the cleavage subunits.\",\n      \"evidence\": \"reconstitution of recombinant CPSF subcomplex and AAUAAA-dependent polyadenylation assay\",\n      \"pmids\": [\"25301781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address how this subcomplex couples to the catalytic cleavage module in vivo\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the structural mechanism of PAS recognition, showing CPSF160 scaffolds WDR33 and CPSF30 and that U3/A6 form a Hoogsteen base pair read by WDR33, and identified the WDR33 KR-rich motif as the specificity determinant.\",\n      \"evidence\": \"3.4 Å cryo-EM of CPSF160–WDR33–CPSF30–AAUAAA, plus crystal structure, XL-MS and fluorescence anisotropy binding assays\",\n      \"pmids\": [\"29208711\", \"29274231\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not quantify the absolute affinity contribution of each subunit\", \"Did not capture the assembled cleavage-competent holo-complex\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Quantified the cooperativity established structurally, showing the ternary CPSF160–WDR33–CPSF30 complex binds AAUAAA at ~3 nM whereas binary complexes are much weaker, demonstrating that both WDR33 and CPSF30 are required for high-affinity recognition.\",\n      \"evidence\": \"fluorescence polarization binding assays with purified proteins and systematic mutagenesis\",\n      \"pmids\": [\"31462423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address kinetics or signal-variant discrimination in vivo\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected the WDR33 recognition module to catalysis by showing RBBP6 binds WDR33 and CPSF73 and activates CPSF endonuclease activity through RNA-dependent recruitment.\",\n      \"evidence\": \"biochemical reconstitution of endonuclease activity and mutational analysis of RBBP6 interactions\",\n      \"pmids\": [\"35177536\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"WDR33–RBBP6 interaction inferred from mutational/sequence analysis rather than direct structure\", \"Structural basis of endonuclease activation not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Explained how WDR33-containing mPSF accommodates the variant AUUAAA signal, showing CPSF30 ZF2 adapts via U2 hydrogen bonds while WDR33 contacts remain unchanged.\",\n      \"evidence\": \"cryo-EM of human mPSF (CPSF160–WDR33–CPSF30–Fip1) with AUUAAA\",\n      \"pmids\": [\"36130077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not survey the full spectrum of non-canonical signals\", \"Functional consequences for variant-signal genes not assessed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended WDR33 substrate scope beyond Pol II mRNAs by showing it and CPSF30 polyadenylate Pol III-derived SINE transcripts in an AAUAAA-dependent manner without the cleavage subcomplex.\",\n      \"evidence\": \"siRNA knockdown of CPSF components and Northern hybridization in HeLa cells\",\n      \"pmids\": [\"39707854\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of mPSF recruitment to Pol III transcripts unclear\", \"Biological role of SINE polyadenylation not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a polyadenylation-independent function by showing alternatively polyadenylated WDR33 isoforms V2/V3 localize to the ER, bind STING, and modulate interferon-β induction and autophagy, with isoform production controlled by CFIm25 and intronic splicing.\",\n      \"evidence\": \"subcellular fractionation, Co-IP, isoform-specific knockdown/overexpression, IFN-β reporter and autophagy assays, plus CFIm25 manipulation and splicing/PA-strength assays\",\n      \"pmids\": [\"38430516\", \"39327832\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional study without independent replication\", \"Direct structural basis of WDR33–STING interaction unknown\", \"Physiological relevance in vivo not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How WDR33's canonical 3'-processing role is integrated with its non-canonical ER/STING isoform functions, and what governs its testis- and stimulation-dependent expression in physiology, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No in vivo genetic model linking WDR33 to organismal phenotype in the corpus\", \"Relationship between nuclear polyadenylation and immune isoform functions uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1, 3, 4, 5, 7]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [2, 6, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 2, 3, 5]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"complexes\": [\"mPSF (CPSF160–WDR33–CPSF30–Fip1)\", \"CPSF\"],\n    \"partners\": [\"CPSF160\", \"CPSF30\", \"FIP1L1\", \"RBBP6\", \"CPSF73\", \"STING1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}