{"gene":"TENT2","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2004,"finding":"Mammalian GLD-2 homologs (human and mouse, later designated TENT2/PAPD4) are cytoplasmic poly(A) polymerases; when tethered to mRNAs in Xenopus oocytes they add poly(A) and stimulate translation, demonstrating that recruitment to RNA is sufficient for polyadenylation activity.","method":"Tethered-function assay in Xenopus oocytes (MS2 coat protein tethering), in vitro poly(A) polymerase assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay combined with functional tethering assay in Xenopus oocytes; foundational paper replicated across subsequent studies","pmids":["15070731"],"is_preprint":false},{"year":2005,"finding":"Vertebrate GLD-2 (TENT2) possesses poly(A) polymerase activity in vivo and in vitro and physically interacts with the cytoplasmic polyadenylation factor CPEB in a conserved manner; it is expressed in the brain in regions associated with synaptic plasticity.","method":"In vitro PAP assay, co-immunoprecipitation with CPEB, in situ hybridization and immunohistochemistry","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic activity plus reciprocal protein interaction (Co-IP with CPEB), single lab but multiple orthogonal methods","pmids":["15987818"],"is_preprint":false},{"year":2012,"finding":"Gld2 (TENT2) directly monoadenylates specific miRNA populations in human fibroblasts and this 3' monoadenylation stabilizes those miRNAs; sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3' end.","method":"Biochemical characterization in human fibroblasts, Northern blot, in vitro adenylation assays, miRNA stability measurements after Gld2 depletion","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct in vitro adenylation assay combined with loss-of-function in human cells and multiple readouts; single lab but orthogonal methods","pmids":["23200856"],"is_preprint":false},{"year":2016,"finding":"The STAR-family RNA-binding protein QKI-7 recruits PAPD4 (TENT2) through its unique carboxyl-terminal region to promote cytoplasmic polyadenylation and translational activation of specific target mRNAs (hnRNPA1, p27kip1, β-catenin) in somatic cells; this polyadenylation of p27kip1 is induced by an anti-mitogenic signal.","method":"Co-immunoprecipitation, tethering assays, transcriptional pulse-chase analysis with deadenylase suppression, reporter assays, Western blot","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, functional tethering, and pulse-chase polyadenylation assay in somatic cells; multiple orthogonal methods in single lab","pmids":["26926106"],"is_preprint":false},{"year":2016,"finding":"Human Gld2 (TENT2) is a bona fide adenylyltransferase with 83-fold preference for ATP over UTP, displaying promiscuous substrate activity toward miRNA, pre-miRNA, and polyadenylated RNA; apo-Gld2 adds only single nucleotides and processivity requires additional RNA-binding proteins. An active-site histidine insertion converts its specificity from ATP to UTP.","method":"In vitro biochemical characterization with purified recombinant enzyme, nucleotide preference assays, active-site mutagenesis, phylogenetic analysis","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with active-site mutagenesis; rigorous biochemical characterization of human protein","pmids":["27284165"],"is_preprint":false},{"year":2016,"finding":"HCV core protein specifically inhibits GLD-2 (TENT2) by direct interaction in the cytoplasm, leading to destabilization of miR-122 by reducing its 3'-terminal single-nucleotide tailing; GLD-2 can add any single ribonucleotide without adenylate preference to the miR-122 3' end.","method":"Co-immunoprecipitation, high-throughput small RNA sequencing from liver biopsies and cell lines, in vitro terminal nucleotidyltransferase assay, miRNA stability assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct protein interaction (Co-IP), in vitro enzymatic assay, and functional miRNA stability experiments; multiple orthogonal methods in single study","pmids":["27366906"],"is_preprint":false},{"year":2016,"finding":"Gld2 (TENT2) interacts with CPEB1 to mediate polyadenylation-induced translation in dendrites of cultured hippocampal neurons; depletion of Gld2 from the hippocampus impairs long-term potentiation evoked by theta burst stimulation. However, miRNA monoadenylation by Gld2 in the hippocampus does not detectably affect miRNA stability or animal behavior in knockout mice.","method":"Hippocampal Gld2 knockout mice, electrophysiology (LTP), miRNA monoadenylation measurements, behavioral assays","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined electrophysiological phenotype and miRNA modification assay; negative behavioral result explicitly reported","pmids":["27495319"],"is_preprint":false},{"year":2019,"finding":"QKI-7 interacts with GLD-2 (TENT2) via its C-terminal region and with Argonaute 2 (Ago2) via its QUA2 domain, thereby recruiting GLD-2 to Ago2-associated miR-122; QKI-7 directly promotes GLD-2-mediated 3' adenylation of miR-122 in vitro and stabilizes miR-122.","method":"Co-immunoprecipitation (GLD-2/QKI-7 and QKI-7/Ago2), in vitro adenylation assay, QKI knockdown/overexpression, Northern blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — reciprocal Co-IP plus in vitro adenylation assay demonstrating functional consequence; multiple orthogonal methods in single lab","pmids":["31792053"],"is_preprint":false},{"year":2019,"finding":"Gld2 (TENT2) activity is regulated by site-specific phosphorylation in its disordered N-terminal domain: phosphomimetic substitutions at S62 and S110 increase activity, while phosphomimetic S116 markedly reduces activity. PKA and Akt1 were identified as kinases that phosphorylate Gld2 at S116, abolishing its nucleotide addition activity.","method":"Phosphomimetic mutagenesis, in vitro adenylation assays, mass spectrometry confirmation of phosphorylation in HEK293 cells, kinase assays with PKA and Akt1","journal":"RNA biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site and PTM mutagenesis combined with in vitro reconstitution and MS validation; multiple orthogonal approaches in single study","pmids":["31057087"],"is_preprint":false},{"year":2020,"finding":"Crystal structures of two rodent GLD-2 (TENT2) proteins revealed that mammalian GLD-2 is an intrinsically robust poly(A) polymerase with an extensively positively charged surface; unlike C. elegans GLD-2 which prefers adenosine-rich RNA substrates, mammalian GLD-2 acts on RNA oligonucleotides of various sequences, and structurally resembles TUT7.","method":"X-ray crystallography (crystal structures of rodent GLD-2), in vitro PAP assays with varied RNA substrates, structural comparison","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure determination combined with in vitro enzymatic characterization revealing substrate promiscuity","pmids":["32633758"],"is_preprint":false},{"year":2021,"finding":"DDX6 is a positive regulator of PAPD4 (TENT2)-containing cytoplasmic polyadenylation machinery: DDX6 physically interacts with Ataxin-2, PABPC1, and PAPD4; DDX6 downregulation increases proportion of target mRNAs with short poly(A) tails and reduces their protein expression, phenocopying Ataxin-2 downregulation.","method":"Ataxin-2 interactome by LC-MS/MS, Co-immunoprecipitation (DDX6/Ataxin-2/PAPD4/PABPC1), poly(A) tail length assays, knockdown/overexpression experiments","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — MS-based interactome confirmed by Co-IP, functional knockdown phenotype; single lab, indirect PAPD4 link via complex","pmids":["33756349"],"is_preprint":false},{"year":2021,"finding":"Gld2 (TENT2) activity and RNA specificity are dynamically regulated by the interplay between phosphorylation and RNA-binding protein interactions: binding of QKI-7 to a short peptide in the Gld2 N-terminal domain relieves autoinhibition and can override Akt1-mediated deactivation at S116; phosphorylation at S62 also relieves N-terminal autoinhibitory function.","method":"In vitro adenylation assays, phosphomimetic mutagenesis, binding assays between Gld2 peptides and QKI-7, Akt1 phosphorylation confirmed in cellular context","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic and binding assays with mutagenesis; single lab extending prior phosphorylation work","pmids":["34288801"],"is_preprint":false},{"year":2022,"finding":"TENT2 contributes to guanylation and uridylation (not only adenylation) on mature miRNAs; in TENT2 knockout cells, 3' tailing is selective rather than random. Abolishing adenylation alone has marginal impact on miRNA levels, but TUT4/7-mediated uridylation (not TENT2) is the primary regulator of miRNA abundance for most miRNAs.","method":"Isogenic TENT2/TUT4/TUT7 single and combination knockout HEK293T cells, deep sequencing of miRNA 3' ends, Northern blot, in vitro tailing assays, rescue experiments","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — isogenic KO cell lines with rescue, deep sequencing, in vitro assays, and Northern blot across multiple knockout combinations; rigorous multi-method single study","pmids":["36071058"],"is_preprint":false},{"year":2025,"finding":"TENT2 loss in mouse hippocampus eliminates miRNA monoadenylation but has no detectable effect on mRNA total poly(A) tail length (measured by direct nanopore sequencing); Tent2 KO neurons show increased excitability and downregulation of synaptic transmission transcripts, linking TENT2-dependent miRNA tailing to excitatory/inhibitory balance.","method":"Tent2 knockout mouse, direct nanopore RNA sequencing (poly(A) tail length), electrophysiology, behavioral assays, differential expression analysis","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with electrophysiology, direct nanopore sequencing, and behavioral phenotyping; multiple orthogonal methods in single study","pmids":["40101932"],"is_preprint":false},{"year":2025,"finding":"TENT2 broadly catalyzes monoadenylation of small non-coding RNAs including Pol-III-transcribed RNAs and a subset of snRNAs; TENT2-mediated monoadenylation inhibits post-transcriptional 3' uridine trimming and extension of Pol-III RNAs, and in the case of 7SL RNA prevents La protein association and promotes assembly into cytoplasmic signal recognition particles.","method":"Genome-wide 3' end sequencing of nascent and steady-state sncRNAs in TENT2 KO cells, deep sequencing, functional characterization of 7SL RNA biogenesis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide sequencing in KO cells with functional consequence on 7SL RNA; preprint, single lab, not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"In the absence of HENMT1, TENT2 (together with TUT4) tails 3'-tRNA fragments (3'-tRFs), contributing to their degradation; this tailing by TENT2 is revealed when HENMT1-mediated 2'-O methylation is absent.","method":"HENMT1 knockout system, small RNA sequencing, identification of TENT2-dependent tailing of 3'-tRFs","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, TENT2 role inferred from sequencing data in HENMT1 KO context, limited direct mechanistic characterization of TENT2 specifically","pmids":[],"is_preprint":true}],"current_model":"TENT2 (GLD-2/PAPD4/TUT2) is a cytoplasmic non-canonical poly(A) polymerase and terminal nucleotidyltransferase that functions primarily as an adenylyltransferase (83-fold preference for ATP over UTP): it extends poly(A) tails of specific mRNAs to promote their translation, and monoadenylates the 3' ends of specific miRNAs and small non-coding RNAs to stabilize them and influence their biogenesis; its substrate specificity and activity are controlled by interaction with RNA-binding partner proteins (CPEB, QKI-7, GLD-3/RNP-8 orthologs) that recruit it to target RNAs and can relieve N-terminal autoinhibition, and by site-specific phosphorylation of its N-terminal domain by PKA and Akt1; in the hippocampus, TENT2-dependent miRNA monoadenylation regulates excitatory/inhibitory balance without affecting bulk mRNA poly(A) tail length."},"narrative":{"mechanistic_narrative":"TENT2 (GLD-2/PAPD4/TUT2) is a cytoplasmic non-canonical poly(A) polymerase and terminal nucleotidyltransferase that controls post-transcriptional gene expression by extending or tailing the 3' ends of distinct RNA classes [PMID:15070731, PMID:27284165]. As a poly(A) polymerase it adds adenosines to specific mRNAs and stimulates their translation, and recruitment to an RNA target is sufficient to trigger this activity [PMID:15070731]. Biochemically it is a bona fide adenylyltransferase with an ~83-fold preference for ATP over UTP, but in isolation the apo-enzyme adds only single nucleotides; processive polyadenylation requires accessory RNA-binding proteins, and a single active-site histidine insertion is sufficient to switch its specificity from ATP to UTP [PMID:27284165]. Substrate selection and recruitment are governed by interacting RNA-binding proteins: CPEB/CPEB1 directs cytoplasmic polyadenylation, including dendritic, plasticity-linked translation in hippocampal neurons [PMID:15987818, PMID:27495319], while the STAR-family protein QKI-7 engages TENT2 through a short N-terminal peptide and bridges it to Argonaute 2-associated miR-122, relieving N-terminal autoinhibition and promoting target-specific 3' adenylation [PMID:26926106, PMID:31792053, PMID:34288801]. Beyond mRNA, TENT2 monoadenylates specific miRNAs and broader small non-coding RNAs, a modification that can stabilize the RNA and shape its biogenesis [PMID:23200856, PMID:36071058]. Its activity is further tuned by site-specific phosphorylation of its disordered N-terminal domain, with PKA- and Akt1-mediated phosphorylation of S116 abolishing nucleotide-addition activity and QKI-7 binding able to override this deactivation [PMID:31057087, PMID:34288801]. In vivo, hippocampal TENT2 supports long-term potentiation and, through miRNA monoadenylation rather than bulk mRNA poly(A) tail changes, regulates neuronal excitability and excitatory/inhibitory balance [PMID:27495319, PMID:40101932].","teleology":[{"year":2004,"claim":"Established that mammalian GLD-2 homologs are cytoplasmic poly(A) polymerases whose activity is gated by recruitment to RNA rather than being constitutive, defining the core enzymatic identity of the gene.","evidence":"Tethered-function assay in Xenopus oocytes plus in vitro poly(A) polymerase assay","pmids":["15070731"],"confidence":"High","gaps":["Did not identify endogenous recruiting partners","No structural basis for the activity"]},{"year":2005,"claim":"Connected TENT2 to the cytoplasmic polyadenylation machinery and to the nervous system by showing a conserved physical interaction with CPEB and brain expression in plasticity-associated regions.","evidence":"In vitro PAP assay, Co-IP with CPEB, in situ hybridization and immunohistochemistry","pmids":["15987818"],"confidence":"High","gaps":["Did not demonstrate CPEB-dependent translation of specific endogenous mRNAs","No functional neuronal phenotype shown"]},{"year":2012,"claim":"Extended TENT2 substrates beyond mRNA by showing it monoadenylates specific miRNAs and that this 3' modification stabilizes them, broadening its role to small-RNA metabolism.","evidence":"Loss-of-function in human fibroblasts, Northern blot, in vitro adenylation and miRNA stability assays","pmids":["23200856"],"confidence":"High","gaps":["Did not define what targets miRNA subsets for monoadenylation","Recruiting factors for miRNA tailing unknown"]},{"year":2016,"claim":"Defined the intrinsic enzymology of human TENT2 — an ATP-preferring adenylyltransferase that is distributive alone and requires partner proteins for processivity — establishing why accessory factors are mechanistically essential.","evidence":"In vitro reconstitution with purified recombinant enzyme, nucleotide preference assays, active-site mutagenesis","pmids":["27284165"],"confidence":"High","gaps":["Did not identify all processivity-conferring partners","Physiological substrate spectrum not mapped"]},{"year":2016,"claim":"Identified QKI-7 as a sequence-specific recruiter that brings TENT2 to defined mRNAs (hnRNPA1, p27kip1, β-catenin) and linked this polyadenylation to anti-mitogenic signaling, showing how target specificity is achieved in somatic cells.","evidence":"Co-IP, tethering assays, transcriptional pulse-chase with deadenylase suppression, reporter assays","pmids":["26926106"],"confidence":"High","gaps":["Did not resolve the QKI-7 binding interface on TENT2","Signal transduction upstream of recruitment not detailed"]},{"year":2016,"claim":"Demonstrated a physiological neuronal role: hippocampal TENT2 supports LTP through CPEB1-mediated dendritic polyadenylation, while showing its miRNA monoadenylation did not detectably affect miRNA stability or behavior in KO mice.","evidence":"Hippocampal Gld2 knockout mice, LTP electrophysiology, miRNA monoadenylation and behavioral assays","pmids":["27495319"],"confidence":"Medium","gaps":["Discrepancy between miRNA tailing and lack of stability/behavior effect unresolved","Specific dendritic mRNA targets not enumerated"]},{"year":2016,"claim":"Showed TENT2 is a target for viral subversion: HCV core protein directly binds and inhibits it, destabilizing miR-122 by blocking 3'-terminal single-nucleotide tailing, placing TENT2 in host-virus RNA regulation.","evidence":"Co-IP, small RNA sequencing from liver and cell lines, in vitro nucleotidyltransferase and miRNA stability assays","pmids":["27366906"],"confidence":"High","gaps":["Did not map the TENT2 region bound by HCV core","In vivo consequences for HCV infection not established"]},{"year":2019,"claim":"Revealed phospho-regulation of TENT2 through its disordered N-terminus, with PKA/Akt1 phosphorylation of S116 abolishing activity, linking the enzyme to upstream signaling kinases.","evidence":"Phosphomimetic mutagenesis, in vitro adenylation assays, MS confirmation in HEK293, PKA/Akt1 kinase assays","pmids":["31057087"],"confidence":"High","gaps":["Physiological stimuli that drive S116 phosphorylation not defined","Did not show phospho-control of specific RNA targets in cells"]},{"year":2019,"claim":"Mechanistically connected recruitment and small-RNA tailing by showing QKI-7 bridges TENT2 to Ago2-bound miR-122 via distinct domains, promoting its 3' adenylation and stabilization.","evidence":"Reciprocal Co-IP (GLD-2/QKI-7, QKI-7/Ago2), in vitro adenylation assay, QKI knockdown/overexpression, Northern blot","pmids":["31792053"],"confidence":"High","gaps":["Generality of Ago2-bridging to other miRNAs untested","Structural detail of the ternary complex absent"]},{"year":2020,"claim":"Provided the structural basis of mammalian TENT2 activity, showing a positively charged, intrinsically robust polymerase that acts on diverse RNA sequences and resembles TUT7, explaining its substrate promiscuity.","evidence":"X-ray crystallography of rodent GLD-2, in vitro PAP assays with varied RNA substrates, structural comparison","pmids":["32633758"],"confidence":"High","gaps":["No structure of human enzyme bound to a partner protein","N-terminal autoinhibitory region not resolved"]},{"year":2021,"claim":"Integrated the two regulatory layers by showing QKI-7 binding to an N-terminal TENT2 peptide relieves autoinhibition and overrides Akt1-mediated S116 deactivation, while S62 phosphorylation also relieves autoinhibition.","evidence":"In vitro adenylation and peptide-binding assays, phosphomimetic mutagenesis, cellular Akt1 phosphorylation","pmids":["34288801"],"confidence":"Medium","gaps":["Combinatorial control under physiological signaling untested","Did not show override at endogenous targets in cells"]},{"year":2021,"claim":"Placed TENT2 within a larger cytoplasmic polyadenylation complex by showing DDX6 acts as a positive regulator interacting with Ataxin-2, PABPC1, and PAPD4 to maintain target mRNA poly(A) length and protein output.","evidence":"Ataxin-2 interactome by LC-MS/MS, Co-IP, poly(A) tail length assays, knockdown/overexpression","pmids":["33756349"],"confidence":"Medium","gaps":["PAPD4 link is indirect through the complex","Direct DDX6-TENT2 contact and target mRNA set not defined"]},{"year":2022,"claim":"Recontextualized TENT2's role in miRNA metabolism by showing its tailing is selective and includes guanylation/uridylation, but that abolishing adenylation alone has marginal impact while TUT4/7 uridylation, not TENT2, dominates miRNA abundance control.","evidence":"Isogenic TENT2/TUT4/TUT7 KO HEK293T cells, deep sequencing of 3' ends, Northern blot, in vitro tailing, rescue","pmids":["36071058"],"confidence":"High","gaps":["Functional purpose of selective non-adenosine tailing unclear","Which miRNAs depend on TENT2 physiologically not pinned down"]},{"year":2025,"claim":"Resolved the in vivo neuronal mechanism by showing TENT2 loss eliminates miRNA monoadenylation without altering bulk mRNA poly(A) length, yet increases neuronal excitability and downregulates synaptic transcripts, tying miRNA tailing to excitatory/inhibitory balance.","evidence":"Tent2 KO mouse, direct nanopore poly(A) sequencing, electrophysiology, behavioral and differential-expression analysis","pmids":["40101932"],"confidence":"High","gaps":["Specific monoadenylated miRNAs driving the phenotype not identified","Behavioral consequences remain limited"]},{"year":2025,"claim":"Expanded the substrate repertoire to Pol-III and other small non-coding RNAs, showing TENT2 monoadenylation blocks uridine trimming/extension and, for 7SL RNA, prevents La binding and promotes signal recognition particle assembly.","evidence":"Genome-wide 3' end sequencing of nascent and steady-state sncRNAs in KO cells, functional 7SL RNA analysis (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Recruitment mechanism to Pol-III RNAs unknown","Breadth of physiological consequences untested"]},{"year":2025,"claim":"Implicated TENT2 in tRNA-fragment surveillance, showing that without HENMT1-mediated 2'-O methylation, TENT2 (with TUT4) tails 3'-tRFs to promote their degradation.","evidence":"HENMT1 KO system, small RNA sequencing identifying TENT2-dependent tailing (preprint)","pmids":[],"confidence":"Low","gaps":["Preprint; TENT2 role inferred from sequencing rather than direct reconstitution","Not independently confirmed","Physiological relevance of 3'-tRF tailing unclear"]},{"year":null,"claim":"It remains unknown how TENT2 selects among its many RNA substrate classes in a given cell type and what governs the choice between adenylation, guanylation, and uridylation at specific 3' ends.","evidence":"No timeline study resolves the determinants of substrate-class and nucleotide selectivity in vivo","pmids":[],"confidence":"Low","gaps":["Substrate-partitioning logic across mRNA, miRNA, and sncRNA unknown","Physiological signals dictating nucleotide identity undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,2,4,5,9,12]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,4,9]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[4,9]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,3,12]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[6,13]}],"complexes":[],"partners":["CPEB1","QKI","AGO2","DDX6","PABPC1","ATXN2","AKT1","PRKACA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6PIY7","full_name":"Poly(A) RNA polymerase GLD2","aliases":["PAP-associated domain-containing protein 4","Terminal nucleotidyltransferase 2","Terminal uridylyltransferase 2","TUTase 2"],"length_aa":484,"mass_kda":56.0,"function":"Cytoplasmic poly(A) RNA polymerase that adds successive AMP monomers to the 3'-end of specific RNAs, forming a poly(A) tail (PubMed:15070731, PubMed:31792053). In contrast to the canonical nuclear poly(A) RNA polymerase, it only adds poly(A) to selected cytoplasmic mRNAs (PubMed:15070731). Does not play a role in replication-dependent histone mRNA degradation (PubMed:18172165). Adds a single nucleotide to the 3' end of specific miRNAs, monoadenylation stabilizes and prolongs the activity of some but not all miRNAs (PubMed:23200856, PubMed:31792053)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q6PIY7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TENT2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"HNRNPH1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TENT2","total_profiled":1310},"omim":[{"mim_id":"614121","title":"TERMINAL NUCLEOTIDYLTRANSFERASE 2; TENT2","url":"https://www.omim.org/entry/614121"},{"mim_id":"604683","title":"KINESIN FAMILY MEMBER 3A; KIF3A","url":"https://www.omim.org/entry/604683"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TENT2"},"hgnc":{"alias_symbol":["FLJ38499","GLD2","TUT2"],"prev_symbol":["PAPD4"]},"alphafold":{"accession":"Q6PIY7","domains":[{"cath_id":"1.10.1410.10","chopping":"154-481","consensus_level":"medium","plddt":94.8035,"start":154,"end":481}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PIY7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PIY7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PIY7-F1-predicted_aligned_error_v6.png","plddt_mean":76.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TENT2","jax_strain_url":"https://www.jax.org/strain/search?query=TENT2"},"sequence":{"accession":"Q6PIY7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6PIY7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6PIY7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PIY7"}},"corpus_meta":[{"pmid":"15070731","id":"PMC_15070731","title":"Mammalian 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mouse, later designated TENT2/PAPD4) are cytoplasmic poly(A) polymerases; when tethered to mRNAs in Xenopus oocytes they add poly(A) and stimulate translation, demonstrating that recruitment to RNA is sufficient for polyadenylation activity.\",\n      \"method\": \"Tethered-function assay in Xenopus oocytes (MS2 coat protein tethering), in vitro poly(A) polymerase assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay combined with functional tethering assay in Xenopus oocytes; foundational paper replicated across subsequent studies\",\n      \"pmids\": [\"15070731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Vertebrate GLD-2 (TENT2) possesses poly(A) polymerase activity in vivo and in vitro and physically interacts with the cytoplasmic polyadenylation factor CPEB in a conserved manner; it is expressed in the brain in regions associated with synaptic plasticity.\",\n      \"method\": \"In vitro PAP assay, co-immunoprecipitation with CPEB, in situ hybridization and immunohistochemistry\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic activity plus reciprocal protein interaction (Co-IP with CPEB), single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15987818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Gld2 (TENT2) directly monoadenylates specific miRNA populations in human fibroblasts and this 3' monoadenylation stabilizes those miRNAs; sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3' end.\",\n      \"method\": \"Biochemical characterization in human fibroblasts, Northern blot, in vitro adenylation assays, miRNA stability measurements after Gld2 depletion\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct in vitro adenylation assay combined with loss-of-function in human cells and multiple readouts; single lab but orthogonal methods\",\n      \"pmids\": [\"23200856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The STAR-family RNA-binding protein QKI-7 recruits PAPD4 (TENT2) through its unique carboxyl-terminal region to promote cytoplasmic polyadenylation and translational activation of specific target mRNAs (hnRNPA1, p27kip1, β-catenin) in somatic cells; this polyadenylation of p27kip1 is induced by an anti-mitogenic signal.\",\n      \"method\": \"Co-immunoprecipitation, tethering assays, transcriptional pulse-chase analysis with deadenylase suppression, reporter assays, Western blot\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, functional tethering, and pulse-chase polyadenylation assay in somatic cells; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"26926106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Human Gld2 (TENT2) is a bona fide adenylyltransferase with 83-fold preference for ATP over UTP, displaying promiscuous substrate activity toward miRNA, pre-miRNA, and polyadenylated RNA; apo-Gld2 adds only single nucleotides and processivity requires additional RNA-binding proteins. An active-site histidine insertion converts its specificity from ATP to UTP.\",\n      \"method\": \"In vitro biochemical characterization with purified recombinant enzyme, nucleotide preference assays, active-site mutagenesis, phylogenetic analysis\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with active-site mutagenesis; rigorous biochemical characterization of human protein\",\n      \"pmids\": [\"27284165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HCV core protein specifically inhibits GLD-2 (TENT2) by direct interaction in the cytoplasm, leading to destabilization of miR-122 by reducing its 3'-terminal single-nucleotide tailing; GLD-2 can add any single ribonucleotide without adenylate preference to the miR-122 3' end.\",\n      \"method\": \"Co-immunoprecipitation, high-throughput small RNA sequencing from liver biopsies and cell lines, in vitro terminal nucleotidyltransferase assay, miRNA stability assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct protein interaction (Co-IP), in vitro enzymatic assay, and functional miRNA stability experiments; multiple orthogonal methods in single study\",\n      \"pmids\": [\"27366906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Gld2 (TENT2) interacts with CPEB1 to mediate polyadenylation-induced translation in dendrites of cultured hippocampal neurons; depletion of Gld2 from the hippocampus impairs long-term potentiation evoked by theta burst stimulation. However, miRNA monoadenylation by Gld2 in the hippocampus does not detectably affect miRNA stability or animal behavior in knockout mice.\",\n      \"method\": \"Hippocampal Gld2 knockout mice, electrophysiology (LTP), miRNA monoadenylation measurements, behavioral assays\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined electrophysiological phenotype and miRNA modification assay; negative behavioral result explicitly reported\",\n      \"pmids\": [\"27495319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"QKI-7 interacts with GLD-2 (TENT2) via its C-terminal region and with Argonaute 2 (Ago2) via its QUA2 domain, thereby recruiting GLD-2 to Ago2-associated miR-122; QKI-7 directly promotes GLD-2-mediated 3' adenylation of miR-122 in vitro and stabilizes miR-122.\",\n      \"method\": \"Co-immunoprecipitation (GLD-2/QKI-7 and QKI-7/Ago2), in vitro adenylation assay, QKI knockdown/overexpression, Northern blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — reciprocal Co-IP plus in vitro adenylation assay demonstrating functional consequence; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"31792053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Gld2 (TENT2) activity is regulated by site-specific phosphorylation in its disordered N-terminal domain: phosphomimetic substitutions at S62 and S110 increase activity, while phosphomimetic S116 markedly reduces activity. PKA and Akt1 were identified as kinases that phosphorylate Gld2 at S116, abolishing its nucleotide addition activity.\",\n      \"method\": \"Phosphomimetic mutagenesis, in vitro adenylation assays, mass spectrometry confirmation of phosphorylation in HEK293 cells, kinase assays with PKA and Akt1\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site and PTM mutagenesis combined with in vitro reconstitution and MS validation; multiple orthogonal approaches in single study\",\n      \"pmids\": [\"31057087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of two rodent GLD-2 (TENT2) proteins revealed that mammalian GLD-2 is an intrinsically robust poly(A) polymerase with an extensively positively charged surface; unlike C. elegans GLD-2 which prefers adenosine-rich RNA substrates, mammalian GLD-2 acts on RNA oligonucleotides of various sequences, and structurally resembles TUT7.\",\n      \"method\": \"X-ray crystallography (crystal structures of rodent GLD-2), in vitro PAP assays with varied RNA substrates, structural comparison\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determination combined with in vitro enzymatic characterization revealing substrate promiscuity\",\n      \"pmids\": [\"32633758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DDX6 is a positive regulator of PAPD4 (TENT2)-containing cytoplasmic polyadenylation machinery: DDX6 physically interacts with Ataxin-2, PABPC1, and PAPD4; DDX6 downregulation increases proportion of target mRNAs with short poly(A) tails and reduces their protein expression, phenocopying Ataxin-2 downregulation.\",\n      \"method\": \"Ataxin-2 interactome by LC-MS/MS, Co-immunoprecipitation (DDX6/Ataxin-2/PAPD4/PABPC1), poly(A) tail length assays, knockdown/overexpression experiments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — MS-based interactome confirmed by Co-IP, functional knockdown phenotype; single lab, indirect PAPD4 link via complex\",\n      \"pmids\": [\"33756349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Gld2 (TENT2) activity and RNA specificity are dynamically regulated by the interplay between phosphorylation and RNA-binding protein interactions: binding of QKI-7 to a short peptide in the Gld2 N-terminal domain relieves autoinhibition and can override Akt1-mediated deactivation at S116; phosphorylation at S62 also relieves N-terminal autoinhibitory function.\",\n      \"method\": \"In vitro adenylation assays, phosphomimetic mutagenesis, binding assays between Gld2 peptides and QKI-7, Akt1 phosphorylation confirmed in cellular context\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic and binding assays with mutagenesis; single lab extending prior phosphorylation work\",\n      \"pmids\": [\"34288801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TENT2 contributes to guanylation and uridylation (not only adenylation) on mature miRNAs; in TENT2 knockout cells, 3' tailing is selective rather than random. Abolishing adenylation alone has marginal impact on miRNA levels, but TUT4/7-mediated uridylation (not TENT2) is the primary regulator of miRNA abundance for most miRNAs.\",\n      \"method\": \"Isogenic TENT2/TUT4/TUT7 single and combination knockout HEK293T cells, deep sequencing of miRNA 3' ends, Northern blot, in vitro tailing assays, rescue experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — isogenic KO cell lines with rescue, deep sequencing, in vitro assays, and Northern blot across multiple knockout combinations; rigorous multi-method single study\",\n      \"pmids\": [\"36071058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TENT2 loss in mouse hippocampus eliminates miRNA monoadenylation but has no detectable effect on mRNA total poly(A) tail length (measured by direct nanopore sequencing); Tent2 KO neurons show increased excitability and downregulation of synaptic transmission transcripts, linking TENT2-dependent miRNA tailing to excitatory/inhibitory balance.\",\n      \"method\": \"Tent2 knockout mouse, direct nanopore RNA sequencing (poly(A) tail length), electrophysiology, behavioral assays, differential expression analysis\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with electrophysiology, direct nanopore sequencing, and behavioral phenotyping; multiple orthogonal methods in single study\",\n      \"pmids\": [\"40101932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TENT2 broadly catalyzes monoadenylation of small non-coding RNAs including Pol-III-transcribed RNAs and a subset of snRNAs; TENT2-mediated monoadenylation inhibits post-transcriptional 3' uridine trimming and extension of Pol-III RNAs, and in the case of 7SL RNA prevents La protein association and promotes assembly into cytoplasmic signal recognition particles.\",\n      \"method\": \"Genome-wide 3' end sequencing of nascent and steady-state sncRNAs in TENT2 KO cells, deep sequencing, functional characterization of 7SL RNA biogenesis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide sequencing in KO cells with functional consequence on 7SL RNA; preprint, single lab, not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In the absence of HENMT1, TENT2 (together with TUT4) tails 3'-tRNA fragments (3'-tRFs), contributing to their degradation; this tailing by TENT2 is revealed when HENMT1-mediated 2'-O methylation is absent.\",\n      \"method\": \"HENMT1 knockout system, small RNA sequencing, identification of TENT2-dependent tailing of 3'-tRFs\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, TENT2 role inferred from sequencing data in HENMT1 KO context, limited direct mechanistic characterization of TENT2 specifically\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TENT2 (GLD-2/PAPD4/TUT2) is a cytoplasmic non-canonical poly(A) polymerase and terminal nucleotidyltransferase that functions primarily as an adenylyltransferase (83-fold preference for ATP over UTP): it extends poly(A) tails of specific mRNAs to promote their translation, and monoadenylates the 3' ends of specific miRNAs and small non-coding RNAs to stabilize them and influence their biogenesis; its substrate specificity and activity are controlled by interaction with RNA-binding partner proteins (CPEB, QKI-7, GLD-3/RNP-8 orthologs) that recruit it to target RNAs and can relieve N-terminal autoinhibition, and by site-specific phosphorylation of its N-terminal domain by PKA and Akt1; in the hippocampus, TENT2-dependent miRNA monoadenylation regulates excitatory/inhibitory balance without affecting bulk mRNA poly(A) tail length.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TENT2 (GLD-2/PAPD4/TUT2) is a cytoplasmic non-canonical poly(A) polymerase and terminal nucleotidyltransferase that controls post-transcriptional gene expression by extending or tailing the 3' ends of distinct RNA classes [#0, #4]. As a poly(A) polymerase it adds adenosines to specific mRNAs and stimulates their translation, and recruitment to an RNA target is sufficient to trigger this activity [#0]. Biochemically it is a bona fide adenylyltransferase with an ~83-fold preference for ATP over UTP, but in isolation the apo-enzyme adds only single nucleotides; processive polyadenylation requires accessory RNA-binding proteins, and a single active-site histidine insertion is sufficient to switch its specificity from ATP to UTP [#4]. Substrate selection and recruitment are governed by interacting RNA-binding proteins: CPEB/CPEB1 directs cytoplasmic polyadenylation, including dendritic, plasticity-linked translation in hippocampal neurons [#1, #6], while the STAR-family protein QKI-7 engages TENT2 through a short N-terminal peptide and bridges it to Argonaute 2-associated miR-122, relieving N-terminal autoinhibition and promoting target-specific 3' adenylation [#3, #7, #11]. Beyond mRNA, TENT2 monoadenylates specific miRNAs and broader small non-coding RNAs, a modification that can stabilize the RNA and shape its biogenesis [#2, #12]. Its activity is further tuned by site-specific phosphorylation of its disordered N-terminal domain, with PKA- and Akt1-mediated phosphorylation of S116 abolishing nucleotide-addition activity and QKI-7 binding able to override this deactivation [#8, #11]. In vivo, hippocampal TENT2 supports long-term potentiation and, through miRNA monoadenylation rather than bulk mRNA poly(A) tail changes, regulates neuronal excitability and excitatory/inhibitory balance [#6, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that mammalian GLD-2 homologs are cytoplasmic poly(A) polymerases whose activity is gated by recruitment to RNA rather than being constitutive, defining the core enzymatic identity of the gene.\",\n      \"evidence\": \"Tethered-function assay in Xenopus oocytes plus in vitro poly(A) polymerase assay\",\n      \"pmids\": [\"15070731\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify endogenous recruiting partners\", \"No structural basis for the activity\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Connected TENT2 to the cytoplasmic polyadenylation machinery and to the nervous system by showing a conserved physical interaction with CPEB and brain expression in plasticity-associated regions.\",\n      \"evidence\": \"In vitro PAP assay, Co-IP with CPEB, in situ hybridization and immunohistochemistry\",\n      \"pmids\": [\"15987818\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not demonstrate CPEB-dependent translation of specific endogenous mRNAs\", \"No functional neuronal phenotype shown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended TENT2 substrates beyond mRNA by showing it monoadenylates specific miRNAs and that this 3' modification stabilizes them, broadening its role to small-RNA metabolism.\",\n      \"evidence\": \"Loss-of-function in human fibroblasts, Northern blot, in vitro adenylation and miRNA stability assays\",\n      \"pmids\": [\"23200856\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define what targets miRNA subsets for monoadenylation\", \"Recruiting factors for miRNA tailing unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the intrinsic enzymology of human TENT2 — an ATP-preferring adenylyltransferase that is distributive alone and requires partner proteins for processivity — establishing why accessory factors are mechanistically essential.\",\n      \"evidence\": \"In vitro reconstitution with purified recombinant enzyme, nucleotide preference assays, active-site mutagenesis\",\n      \"pmids\": [\"27284165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify all processivity-conferring partners\", \"Physiological substrate spectrum not mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified QKI-7 as a sequence-specific recruiter that brings TENT2 to defined mRNAs (hnRNPA1, p27kip1, β-catenin) and linked this polyadenylation to anti-mitogenic signaling, showing how target specificity is achieved in somatic cells.\",\n      \"evidence\": \"Co-IP, tethering assays, transcriptional pulse-chase with deadenylase suppression, reporter assays\",\n      \"pmids\": [\"26926106\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the QKI-7 binding interface on TENT2\", \"Signal transduction upstream of recruitment not detailed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated a physiological neuronal role: hippocampal TENT2 supports LTP through CPEB1-mediated dendritic polyadenylation, while showing its miRNA monoadenylation did not detectably affect miRNA stability or behavior in KO mice.\",\n      \"evidence\": \"Hippocampal Gld2 knockout mice, LTP electrophysiology, miRNA monoadenylation and behavioral assays\",\n      \"pmids\": [\"27495319\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Discrepancy between miRNA tailing and lack of stability/behavior effect unresolved\", \"Specific dendritic mRNA targets not enumerated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed TENT2 is a target for viral subversion: HCV core protein directly binds and inhibits it, destabilizing miR-122 by blocking 3'-terminal single-nucleotide tailing, placing TENT2 in host-virus RNA regulation.\",\n      \"evidence\": \"Co-IP, small RNA sequencing from liver and cell lines, in vitro nucleotidyltransferase and miRNA stability assays\",\n      \"pmids\": [\"27366906\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the TENT2 region bound by HCV core\", \"In vivo consequences for HCV infection not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed phospho-regulation of TENT2 through its disordered N-terminus, with PKA/Akt1 phosphorylation of S116 abolishing activity, linking the enzyme to upstream signaling kinases.\",\n      \"evidence\": \"Phosphomimetic mutagenesis, in vitro adenylation assays, MS confirmation in HEK293, PKA/Akt1 kinase assays\",\n      \"pmids\": [\"31057087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological stimuli that drive S116 phosphorylation not defined\", \"Did not show phospho-control of specific RNA targets in cells\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mechanistically connected recruitment and small-RNA tailing by showing QKI-7 bridges TENT2 to Ago2-bound miR-122 via distinct domains, promoting its 3' adenylation and stabilization.\",\n      \"evidence\": \"Reciprocal Co-IP (GLD-2/QKI-7, QKI-7/Ago2), in vitro adenylation assay, QKI knockdown/overexpression, Northern blot\",\n      \"pmids\": [\"31792053\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of Ago2-bridging to other miRNAs untested\", \"Structural detail of the ternary complex absent\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural basis of mammalian TENT2 activity, showing a positively charged, intrinsically robust polymerase that acts on diverse RNA sequences and resembles TUT7, explaining its substrate promiscuity.\",\n      \"evidence\": \"X-ray crystallography of rodent GLD-2, in vitro PAP assays with varied RNA substrates, structural comparison\",\n      \"pmids\": [\"32633758\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of human enzyme bound to a partner protein\", \"N-terminal autoinhibitory region not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Integrated the two regulatory layers by showing QKI-7 binding to an N-terminal TENT2 peptide relieves autoinhibition and overrides Akt1-mediated S116 deactivation, while S62 phosphorylation also relieves autoinhibition.\",\n      \"evidence\": \"In vitro adenylation and peptide-binding assays, phosphomimetic mutagenesis, cellular Akt1 phosphorylation\",\n      \"pmids\": [\"34288801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Combinatorial control under physiological signaling untested\", \"Did not show override at endogenous targets in cells\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed TENT2 within a larger cytoplasmic polyadenylation complex by showing DDX6 acts as a positive regulator interacting with Ataxin-2, PABPC1, and PAPD4 to maintain target mRNA poly(A) length and protein output.\",\n      \"evidence\": \"Ataxin-2 interactome by LC-MS/MS, Co-IP, poly(A) tail length assays, knockdown/overexpression\",\n      \"pmids\": [\"33756349\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PAPD4 link is indirect through the complex\", \"Direct DDX6-TENT2 contact and target mRNA set not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Recontextualized TENT2's role in miRNA metabolism by showing its tailing is selective and includes guanylation/uridylation, but that abolishing adenylation alone has marginal impact while TUT4/7 uridylation, not TENT2, dominates miRNA abundance control.\",\n      \"evidence\": \"Isogenic TENT2/TUT4/TUT7 KO HEK293T cells, deep sequencing of 3' ends, Northern blot, in vitro tailing, rescue\",\n      \"pmids\": [\"36071058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional purpose of selective non-adenosine tailing unclear\", \"Which miRNAs depend on TENT2 physiologically not pinned down\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the in vivo neuronal mechanism by showing TENT2 loss eliminates miRNA monoadenylation without altering bulk mRNA poly(A) length, yet increases neuronal excitability and downregulates synaptic transcripts, tying miRNA tailing to excitatory/inhibitory balance.\",\n      \"evidence\": \"Tent2 KO mouse, direct nanopore poly(A) sequencing, electrophysiology, behavioral and differential-expression analysis\",\n      \"pmids\": [\"40101932\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific monoadenylated miRNAs driving the phenotype not identified\", \"Behavioral consequences remain limited\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded the substrate repertoire to Pol-III and other small non-coding RNAs, showing TENT2 monoadenylation blocks uridine trimming/extension and, for 7SL RNA, prevents La binding and promotes signal recognition particle assembly.\",\n      \"evidence\": \"Genome-wide 3' end sequencing of nascent and steady-state sncRNAs in KO cells, functional 7SL RNA analysis (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Recruitment mechanism to Pol-III RNAs unknown\", \"Breadth of physiological consequences untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated TENT2 in tRNA-fragment surveillance, showing that without HENMT1-mediated 2'-O methylation, TENT2 (with TUT4) tails 3'-tRFs to promote their degradation.\",\n      \"evidence\": \"HENMT1 KO system, small RNA sequencing identifying TENT2-dependent tailing (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint; TENT2 role inferred from sequencing rather than direct reconstitution\", \"Not independently confirmed\", \"Physiological relevance of 3'-tRF tailing unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how TENT2 selects among its many RNA substrate classes in a given cell type and what governs the choice between adenylation, guanylation, and uridylation at specific 3' ends.\",\n      \"evidence\": \"No timeline study resolves the determinants of substrate-class and nucleotide selectivity in vivo\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Substrate-partitioning logic across mRNA, miRNA, and sncRNA unknown\", \"Physiological signals dictating nucleotide identity undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 2, 4, 5, 9, 12]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 4, 9]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [4, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 3, 12]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [6, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CPEB1\", \"QKI\", \"AGO2\", \"DDX6\", \"PABPC1\", \"ATXN2\", \"AKT1\", \"PRKACA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}