Affinage

TUT4

Terminal uridylyltransferase 4 · UniProt Q5TAX3

Length
1644 aa
Mass
185.2 kDa
Annotated
2026-04-28
25 papers in source corpus 20 papers cited in narrative 20 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

TUT4 (ZCCHC11) is a noncanonical terminal uridylyl transferase that adds non-templated uridine residues to the 3′ ends of diverse RNA substrates—including pre-miRNAs, mature miRNAs, histone mRNAs, LINE-1 mRNAs, and viral RNAs—thereby governing RNA stability, miRNA biogenesis, retrotransposon restriction, and antiviral defense. In its best-characterized role, Lin28 recruits TUT4 to pre-let-7 via a GGAG motif in the terminal loop; a cryo-EM-resolved ternary complex shows that the N-terminal Lin28-interacting module (LIM) captures Lin28-bound pre-let-7, followed by association of the C-terminal catalytic module (CM), which unwinds the stem and processively oligouridylates the 3′ end, blocking Dicer processing (PMID:19703396, PMID:28671666, PMID:41521656). TUT4 follows an ordered kinetic mechanism in which UTP binds before RNA, with selectivity for UTP encoded primarily in Km, and preferentially uridylates single-stranded 3′ termini; a ZnF2 domain with two distinct RNA-binding surfaces contributes to substrate diversity across miRNA, mRNA, and retrotransposon targets (PMID:35797480, PMID:34719327, PMID:30122351). TUT4 is the dominant enzyme for miRNA uridylation in cells, and its loss leads to compensatory 3′ adenylation of isomiRs, dysregulation of specific miRNAs including let-7, and downstream signaling changes such as altered AKT phosphorylation and IGF-1 expression (PMID:36071058, PMID:23209448).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 2006 Medium

    Before its enzymatic activity was characterized, TUT4 was identified as a cytoplasmic/nuclear shuttling protein that interacts with TIFA and negatively regulates TLR-mediated NF-κB signaling, placing it in innate immune signaling pathways.

    Evidence GST pulldown, AP-MS, NF-κB reporter assay, subcellular fractionation in LPS-stimulated cells

    PMID:16643855

    Open questions at the time
    • NF-κB regulatory activity not linked to uridylation — catalytic mechanism unknown at this stage
    • TIFA interaction confirmed only by pulldown without reciprocal validation
    • Whether NF-κB suppression is direct or indirect remains undefined
  2. 2009 High

    Three independent studies simultaneously established TUT4 as the uridylyl transferase that oligouridylates pre-let-7 (Lin28-dependent, blocking Dicer) and uridylates mature miR-26a (relieving IL-6 silencing), defining its core enzymatic identity as a UTP-dependent terminal transferase acting on both precursor and mature miRNAs.

    Evidence In vitro uridylation reconstitution, knockdown in mESCs and human cells, catalytically inactive mutants, small RNA 3′-end sequencing

    PMID:19701194 PMID:19703396 PMID:19713958

    Open questions at the time
    • Domain architecture responsible for Lin28 interaction and substrate selectivity not yet mapped
    • Whether other TUTases contribute redundantly was unresolved
    • Structural basis of the Lin28–TUT4–pre-let-7 ternary complex unknown
  3. 2010 High

    TUT4's substrate repertoire was extended beyond miRNAs when it was shown to uridylate replication-dependent histone mRNAs for degradation at the end of S-phase, establishing TUT4 as a general mRNA-decay-promoting uridylase.

    Evidence siRNA knockdown with RT-PCR quantification of uridylated histone mRNAs, comparison with PAPD1/PAPD5 knockdowns

    PMID:21051505

    Open questions at the time
    • How TUT4 is recruited to histone mRNAs (specific adaptor or RNA feature) was not defined
    • Contribution of TUT7 to histone mRNA uridylation not assessed
  4. 2011 Medium

    TUT4 was found to promote G1-to-S cell cycle progression through upregulation of cyclins D1/A and CDK4 via its N-terminal region, independent of uridyltransferase catalytic activity, revealing a separable non-enzymatic function.

    Evidence Overexpression/knockdown, catalytically inactive mutant and N-terminal truncation constructs, cell cycle analysis

    PMID:22006926

    Open questions at the time
    • Molecular target of the N-terminal proliferative activity not identified
    • Single-lab observation not independently replicated
    • Whether this contributes to tumorigenesis in vivo is untested
  5. 2012 High

    Domain mapping revealed that a single C2H2 zinc finger mediates TUT4's functional interaction with Lin28, and TUT7 was identified as a redundant partner for let-7 control in ESCs; a knockout mouse showed TUT4 performs pervasive miRNA uridylation in vivo, modulating IGF-1 expression and postnatal growth.

    Evidence Domain deletion/mutagenesis with reconstitution assays, Zcchc11-knockout mouse with deep small RNA sequencing

    PMID:22898984 PMID:23209448

    Open questions at the time
    • Structural basis of the ZnF–Lin28 interaction not resolved
    • Relative in vivo contributions of TUT4 vs TUT7 not fully delineated across tissues
  6. 2014 High

    A bipartite sequence motif on mature miRNAs was identified as necessary and sufficient for TUT4/7-mediated monouridylation, and Trim25 was discovered as an RNA-binding cofactor that activates Lin28/TUT4-dependent oligouridylation, refining the substrate-selection rules.

    Evidence In vitro uridylation with motif mutants, RNA pulldown/MS for Trim25 identification, zebrafish developmental assay

    PMID:25223788 PMID:25457611

    Open questions at the time
    • Trim25 cofactor role confirmed by single lab; independent replication pending
    • How the bipartite motif is recognized structurally was unresolved
  7. 2017 High

    Crystallography of TUT7 CM and biochemical reconstitution revealed the two-module architecture (CM + LIM) that switches between mono- and oligouridylation: Lin28's ZK domain drives ternary complex formation and the ZK2 domain engages the growing oligo(U) tail.

    Evidence Crystal structure of TUT7 CM, domain deletion/mutagenesis, in vitro uridylation reconstitution

    PMID:28671666

    Open questions at the time
    • TUT4-specific structure not yet solved — inferred from TUT7 homology
    • Mechanism of processive elongation vs. distributive addition not fully distinguished
  8. 2018 High

    TUT4/7 were shown to restrict LINE-1 retrotransposition by uridylating L1 mRNA 3′ ends, cooperating with MOV10 to antagonize L1-ORF1p RNA chaperone activity, extending TUT4 function to genome defense.

    Evidence TUT4/7 knockdown in human cells and mouse testes, LINE-1 retrotransposition assay, subcellular fractionation

    PMID:30122351

    Open questions at the time
    • Relative contributions of TUT4 vs TUT7 in LINE-1 restriction in different tissues not fully resolved
    • Whether L1 uridylation triggers specific decay pathway (e.g., DIS3L2) not tested
  9. 2021 Medium

    NMR structural analysis of TUT4/7 ZnF2 revealed two distinct RNA-binding surfaces that engage different nucleobases, providing a structural explanation for how TUT4 recognizes diverse RNA substrates.

    Evidence NMR structure, RNA-binding assays with domain mutants

    PMID:34719327

    Open questions at the time
    • Single-lab NMR study; in vivo contribution of each binding surface not tested
    • How ZnF2 coordinates with the CM during catalysis is unknown
  10. 2022 High

    Rigorous steady-state kinetics established that TUT4 follows an ordered mechanism (UTP binds before RNA), with UTP selectivity encoded in Km and preference for unpaired 3′ ends; genetic knockouts confirmed TUT4 as the dominant miRNA uridylase over TUT7, with loss leading to adenylated isomiR compensation and AKT signaling dysregulation.

    Evidence Steady-state kinetic assays with nucleotide/RNA variants; isogenic HEK293T TUT4/TUT7/TENT2 KO lines with deep sequencing and rescue

    PMID:35797480 PMID:36071058

    Open questions at the time
    • Pre-steady-state kinetics and processivity measurements not performed
    • Which adenylase compensates in TUT4 KO cells not fully defined beyond TENT2
  11. 2023 Medium

    TUT4/7 were implicated in antiviral defense against coronaviruses by uridylating subgenomic RNAs bearing short poly(A) tails, targeting them for decay and limiting viral replication.

    Evidence Splint-ligation poly(A) tail measurement, TUT4/7 siRNA depletion, viral replication quantification in MHV-infected cells

    PMID:37085578

    Open questions at the time
    • Single-lab finding; not tested in SARS-CoV-2 or other coronaviruses
    • Downstream decay pathway (DIS3L2 involvement) not confirmed for viral RNAs
  12. 2024 Medium

    In FOCAD-deleted cancer cells with destabilized SKI complex, TUT7 (but not TUT4) becomes essential for a salvage RNA decay pathway with DIS3L2; pharmacological TUT4/7 inhibition selectively kills FOCAD-deficient cells, revealing a synthetic-lethal therapeutic opportunity.

    Evidence CRISPR KO, FOCAD rescue, TUT4/7 small molecule inhibitors in vitro and in vivo

    PMID:39235218

    Open questions at the time
    • TUT4-specific role in this salvage pathway is minimal — finding is largely TUT7-driven
    • Selectivity profile and off-target effects of TUT4/7 inhibitors not fully characterized
  13. 2026 High

    Cryo-EM of the full human TUT4–Lin28A–oligo(U)-pre-let-7 complex resolved the elongation mechanism: the LIM captures Lin28-bound pre-let-7, the CM associates via protein–protein contacts, the pre-let-7 stem is unwound, and a finger domain clamps the duplex for processive uridine addition.

    Evidence Cryo-EM structure determination, structure-guided mutagenesis, biochemical reconstitution

    PMID:41521656

    Open questions at the time
    • Initiation-to-elongation transition not captured — only elongation state resolved
    • How Trim25 cofactor integrates into the structural complex is unknown
    • Structural basis for mono- vs. oligo-uridylation switch not fully resolved at atomic level for TUT4

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include: (1) how TUT4 is recruited to non-miRNA substrates such as histone mRNAs and LINE-1 mRNAs; (2) the structural mechanism distinguishing monouridylation from oligouridylation in the absence of Lin28; (3) the in vivo significance and selectivity of TUT4's non-enzymatic proliferative activity; and (4) the physiological role of TUT4-mediated uridylation in antiviral defense across different virus families.
  • No structural information on TUT4 in complex with mRNA substrates
  • Non-catalytic cell cycle function has no identified molecular target
  • Relative TUT4 vs TUT7 contributions in most physiological contexts remain tissue- and context-dependent

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 10 GO:0003723 RNA binding 4 GO:0098772 molecular function regulator activity 2
Localization
GO:0005829 cytosol 2 GO:0005634 nucleus 1
Partners
DIS3L2LIN28ALIN28BMOV10TIFATRIM25TUT7
Complex memberships
Lin28–TUT4–pre-let-7 ternary complex

Evidence

Reading pass · 20 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 TUT4 (TUTase4) is the uridylyl transferase responsible for oligouridylation of pre-let-7 microRNA precursors. Lin28 recruits TUT4 to pre-let-7 by recognizing a GGAG tetra-nucleotide sequence motif in the terminal loop; TUT4 then adds an oligouridine tail that blocks Dicer processing. Biochemical identification, knockdown, in vitro uridylation assay Cell High 19703396
2009 Zcchc11 (TUT4) is the 3' terminal uridylyl transferase responsible for Lin28-mediated pre-let-7 uridylation in mouse embryonic stem cells; its activity is UTP-dependent, selective for let-7, and recruited by Lin28. Knockdown or catalytically inactive TUTase relieves inhibition of let-7 processing. Knockdown, overexpression of catalytically inactive mutant, in vitro UTP-dependent uridylation assay, reporter gene assay Nature structural & molecular biology High 19713958
2009 Zcchc11 (TUT4) is a ribonucleotidyltransferase with preference for uridine that uridylates mature miR-26a family members at their 3' ends, thereby abrogating IL-6 repression and maintaining IL-6 mRNA poly(A) tail length and stability. Knockdown, small RNA sequencing of 3' ends, in vitro uridylation assay Nature cell biology High 19701194
2010 Human ZCCHC11 (TUT4) associates with replication-dependent histone mRNAs and is the terminal U-transferase responsible for their 3' uridylation and subsequent degradation following inhibition or completion of DNA replication. Knockdown (siRNA), RT-PCR quantification of uridylated histone mRNAs, selectivity established by comparison with PAPD1/PAPD5 knockdowns RNA (New York, N.Y.) High 21051505
2012 A single C2H2-type zinc finger domain of Zcchc11 (TUT4) is necessary and sufficient for the functional interaction with Lin28, enabling Lin28-enhanced pre-let-7 uridylation. Zcchc6 (TUT7) is an alternative TUTase that functions redundantly with Zcchc11 in embryonic stem cells to control let-7 biogenesis. Biochemical dissection, reconstitution assays, domain deletion/mutagenesis, ESC knockdown RNA (New York, N.Y.) High 22898984
2012 Zcchc11 (TUT4) mediates pervasive 3' terminal uridylation of mature miRNAs in vivo; Zcchc11-deficient mice show decreased terminal uridine frequencies on diverse mature miRNAs without changes in miRNA abundance. This uridylation relieves miRNA silencing of IGF-1 mRNA, enhancing IGF-1 expression and postnatal growth. Zcchc11 knockout mouse, deep sequencing of small RNAs, in vitro uridylation assay, IGF-1 mRNA/protein measurement PLoS genetics High 23209448
2006 ZCCHC11 (TUT4) interacts with TIFA (TRAF-interacting protein with FHA domain), translocates from nucleus to cytoplasm in response to LPS, and functions as a negative regulator of TLR-mediated NF-κB activation. The N-terminal C2H2-type zinc finger region is sufficient for NF-κB suppression. GST pulldown, affinity purification with mass spectrometry, subcellular fractionation/localization, siRNA knockdown, overexpression, NF-κB reporter assay Biochemical and biophysical research communications Medium 16643855
2011 TUT4 (Zcchc11) promotes G1-to-S phase cell cycle progression by upregulating cyclins D1 and A and CDK4, through both Rb-dependent and Rb-independent mechanisms. Remarkably, this proliferative activity resides in the N-terminal region of the protein and does not require uridyltransferase activity. Loss-of-function (knockdown) and gain-of-function (overexpression) experiments, uridyltransferase-inactive point mutant, N-terminal truncation constructs, cell cycle analysis The Journal of biological chemistry Medium 22006926
2014 Zcchc11 (TUT4) and Zcchc6 (TUT7) selectively 3'-monouridylate a specific subset of mature miRNAs involved in cell differentiation and Hox gene control, defined by a bipartite sequence motif necessary and sufficient for catalysis. Loss of TUTase-dependent uridylation is accompanied by a concomitant increase in 3'-monoadenylation. In vitro uridylation assay, sequence motif mapping, deep sequencing of small RNAs after TUTase depletion, zebrafish developmental assay Nucleic acids research High 25223788
2014 Trim25 is an RNA-specific cofactor for Lin28a/TUT4-mediated uridylation. Trim25 binds the conserved terminal loop (CTL) of pre-let-7 and activates TUT4, enabling more efficient and substrate-specific Lin28a-mediated uridylation; without Trim25, TUT4 does not efficiently uridylate pre-let-7. RNA pulldown coupled to quantitative mass spectrometry, co-immunoprecipitation, in vitro uridylation assay Cell reports Medium 25457611
2017 TUT4 and TUT7 utilize two multi-domain functional modules to switch between mono- and oligouridylation of pre-let-7: a catalytic module (CM) essential for both activities and a Lin28-interacting module (LIM) indispensable for oligouridylation. Crystal structure of TUT7 CM trapped in monoU state reveals a duplex-RNA-binding pocket; the ZK domain of Lin28 drives stable ternary complex formation for the oligoU switch, and ZK2 of TUT4(7) engages the growing oligoU tail. Crystal structure (TUT7 CM), domain deletion/mutagenesis, in vitro uridylation reconstitution, biochemical complex formation assays Nature structural & molecular biology High 28671666
2018 TUT4 and TUT7 uridylate LINE-1 mRNA 3' ends to restrict retrotransposition. TUT4 is enriched in cytoplasmic foci and destabilizes LINE-1 mRNAs, while TUT7 acts in the cytoplasm to inhibit reverse transcription of reimported LINE-1 mRNAs. TUT4/7 cooperate with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of L1-ORF1p. TUT4/7 knockdown in human cellular models and mouse testes, LINE-1 retrotransposition assay, subcellular fractionation/localization, co-functional analysis with MOV10 Cell High 30122351
2021 TUT4(7) ZnF2 domain contains two distinct RNA-binding surfaces used in the interaction with different RNA nucleobases in different targets, encoding diversity in TUT4(7) selectivity. Unlike other CCHC ZnFs, ZnF2 acts independently of ZnF3 in miRNA recognition, while ZnF1 has lost intrinsic RNA-binding capability. NMR structural analysis, RNA-binding assays with domain mutants RNA biology Medium 34719327
2022 TUT4 (Zcchc11/Z11) follows a steady-state ordered kinetic mechanism in which UTP binds before RNA; selectivity for UTP over CTP, ATP, and GTP is manifested primarily in Km,XTP; the enzyme preferentially uridylates RNA lacking base-pairing near the 3' terminus; kcat values are similar across substrate sizes but Km,RNA varies with substrate length. Steady-state kinetic assays, substrate specificity studies with ribonucleoside triphosphate variants and truncated RNA substrates Biochemistry High 35797480
2022 TUT4 uridylates most miRNAs in cells, whereas TUT7 is largely dispensable for miRNA uridylation; abolishing uridylation by TUT4/7 knockout dysregulates a specific set of miRNAs and leads to replacement of uridylated isomiRs by adenylated isomiRs. TUT4/7 indirectly regulate AKT phosphorylation via let-7a-mediated control. Isogenic HEK293T knockout cell lines (TENT2, TUT4, TUT7 single and combined KO), deep sequencing, Northern blot, in vitro assays, rescue experiments Nature communications High 36071058
2023 TUT4 and TUT7 uridylate coronavirus (MHV) subgenomic RNAs with short poly(A) tails (<22 nt), marking them for decay; depletion of TUT4/7 increases viral replication capacity and reduces the population of uridylated short-tailed subgenomic RNAs at late infection stages. Splint-ligation poly(A) tail length measurement, TUT4/7 siRNA depletion, viral replication quantification Communications biology Medium 37085578
2024 TUT7 (but not TUT4) is specifically required for viability of FOCAD-deleted cancer cells; in the absence of FOCAD, which disrupts the SKI complex stability, TUT7 and DIS3L2 form a salvage RNA decay mechanism. Pharmacological TUT4/7 inhibition selectively kills FOCAD-deficient cancer cells, and FOCAD reintroduction restores SKI complex and reduces TUT7 dependency. CRISPR knockout, FOCAD rescue experiments, TUT4/7 small molecule inhibitors (in vitro and in vivo antiproliferative assays), public functional genomics data analysis Molecular cancer therapeutics Medium 39235218
2026 Cryo-EM structure of human TUT4 complexed with Lin28A and oligo-uridylated pre-let-7 reveals the elongation-stage mechanism: Lin28A recruits pre-let-7 to the N-terminal LIM via terminal stem-loop interactions; the C-terminal CM then associates with the LIM through protein-protein interactions; the double-stranded stem of pre-let-7 is unwound and the 3' end positioned in the CM catalytic site; during elongation, the CM finger domain clamps the pre-let-7 duplex region for processive uridine tail addition. Cryo-EM structure determination, biochemical reconstitution, structure-guided mutagenesis Nucleic acids research High 41521656
2025 TUT4 uridylates 3'-tRNA fragments (3'-tRFs) in the absence of the methyltransferase HENMT1, contributing to small RNA turnover; HENMT1-mediated 2'-O methylation protects 3'-tRFs from TUT4/TENT2-mediated tailing and degradation. HENMT1 knockout, small RNA sequencing, non-templated tailing quantification bioRxiv (preprint)preprint Low bio_10.1101_2025.05.12.650695
2025 In the ZAP-mediated RNA decay (ZMD) pathway, TUT4/TUT7 uridylate the 5' cleavage fragment generated by KHNYN endonuclease on ZAP-bound viral RNA, and this uridylated fragment is subsequently degraded by DIS3L2. Pathway ordering experiments, RNase-resistant co-immunoprecipitation, RNA decay assays bioRxiv (preprint)preprint Low bio_10.1101_2025.04.28.650959

Source papers

Stage 0 corpus · 25 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2009 TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 664 19703396
2009 Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nature structural & molecular biology 421 19713958
2009 Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nature cell biology 247 19701194
2012 Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7). RNA (New York, N.Y.) 176 22898984
2013 miR-26a enhances miRNA biogenesis by targeting Lin28B and Zcchc11 to suppress tumor growth and metastasis. Oncogene 99 24056962
2014 Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4). Nucleic acids research 85 25223788
2010 The human cytoplasmic RNA terminal U-transferase ZCCHC11 targets histone mRNAs for degradation. RNA (New York, N.Y.) 85 21051505
2018 Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s. Cell 73 30122351
2014 Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation. Cell reports 71 25457611
2012 Zcchc11 uridylates mature miRNAs to enhance neonatal IGF-1 expression, growth, and survival. PLoS genetics 46 23209448
2017 Multi-domain utilization by TUT4 and TUT7 in control of let-7 biogenesis. Nature structural & molecular biology 45 28671666
2015 Identification of small molecule inhibitors of Zcchc11 TUTase activity. RNA biology 43 26114892
2022 TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance. Nature communications 42 36071058
2006 A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling. Biochemical and biophysical research communications 41 16643855
2011 Terminal uridyltransferase enzyme Zcchc11 promotes cell proliferation independent of its uridyltransferase activity. The Journal of biological chemistry 16 22006926
2011 E2F1 and KIAA0191 expression predicts breast cancer patient survival. BMC research notes 14 21453498
2023 TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication. Communications biology 12 37085578
2021 RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell type. RNA (New York, N.Y.) 12 34949722
2022 Terminal Uridylyltransferases TUT4/7 Regulate microRNA and mRNA Homeostasis. Cells 8 36497000
2024 Targeting the Synthetic Lethal Relationship between FOCAD and TUT7 Represents a Potential Therapeutic Opportunity for TUT4/7 Small-Molecule Inhibitors in Cancer. Molecular cancer therapeutics 5 39235218
2022 Differentially Expressed mRNAs and Potential Mechanisms of Radiation-Induced TUT4-/- Esophageal Cell Injury. Dose-response : a publication of International Hormesis Society 3 36324561
2022 Kinetic and Mechanistic Studies of the Terminal Uridylyltransferase, Zcchc11 (TUT4). Biochemistry 2 35797480
2021 The distinct RNA-interaction modes of a small ZnF domain underlay TUT4(7) diverse action in miRNA regulation. RNA biology 1 34719327
2026 Mechanistic insights into Lin28-dependent oligo-uridylylation of pre-let-7 by TUT4. Nucleic acids research 0 41521656
2025 Potential protective regulatory effects on radiation-induced esophageal injury in TUT4-/- mice. Frontiers in oncology 0 40933885