TNPO3

TNPO3 (Transportin-3/TRN-SR2) is a nuclear import receptor of the importin-β family that delivers serine/arginine-rich cargoes through the nuclear pore and serves both gene-expression programs and viral biology (PMID:40360518, PMID:23414560). Structurally, it recognizes non-classical NLSs: crystallography of the TNPO3-CIRBP complex shows that recognition can rely on tyrosine rather than serine/arginine residues and is phosphorylation-independent, with serine/tyrosine phosphorylation within the NLS inhibiting binding (PMID:40360518). Through this import activity TNPO3 sustains tissue- and lineage-specific programs: it transports the SR splicing factor SRSF1 during myogenesis (PMID:33452620), imports the transcription factor EBF1 via its immunoglobulin-like fold domain (critical residue E271) to enable B cell programming under antagonistic Notch signaling (PMID:36167471), and is required for correct splicing of the semi-invariant TCRα pre-mRNA of iNKT cells, a role definitively separated from a developmental one by transgenic rescue with a pre-spliced cDNA (PMID:37339974). In HIV-1 infection, the viral capsid—not integrase—is the dominant determinant of TNPO3 dependence (PMID:19846519, PMID:22145813), and TNPO3 acts chiefly by maintaining the capsid-binding protein CPSF6 in the nucleus; TNPO3 depletion drives CPSF6 into the cytoplasm where it aberrantly stabilizes the CA core and blocks nuclear import, an effect reversed by restoring CPSF6 nuclear localization or co-depleting CPSF6 (PMID:23414560, PMID:23622145). TNPO3 additionally binds the HIV-1 intasome directly through the integrase C-terminal domain (PMID:22872640) and stimulates capsid uncoating in vitro in a RanGTP-sensitive manner (PMID:23097435). Loss-of-function frameshift mutations in TNPO3 cause autosomal dominant limb-girdle muscular dystrophy (LGMD1F/LGMD D2) by producing a C-terminally extended protein that mislocalizes away from nuclear pore complexes (PMID:23667635, PMID:31192305).

1. 2009 High

   Established which viral component dictates the requirement for TNPO3, distinguishing capsid- from integrase-driven dependence and reframing TNPO3 as a capsid-linked host factor.

   Evidence MLV/HIV-1 chimera virus infectivity in TNPO3-knockdown cells with in vitro integrase binding assays

   PMID:19846519

   Open questions at the time

   • Did not define the molecular intermediate connecting capsid to TNPO3
   • Mechanism of how capsid sensitizes infection to TNPO3 unresolved
2. 2011 High

   Localized TNPO3's pro-viral action to a post-nuclear-entry step and confirmed capsid as the genetic determinant via systematic mutagenesis, narrowing where in the lifecycle TNPO3 acts.

   Evidence Panel of 27 CA mutant single-cycle vectors with step-specific qPCR readouts across multiple cell types; IN mutant binding/import assays

   PMID:22145813 PMID:22176773

   Open questions at the time

   • Did not identify the downstream effector mediating the post-entry block
   • Negative IN-import finding from a single lab
3. 2012 High

   Provided biochemical and biophysical characterization of TNPO3, including direct intasome binding and in vitro stimulation of capsid uncoating, defining candidate direct molecular activities.

   Evidence In vitro uncoating assays with purified TNPO3/CypA and RanGTP; CD, AUC, SAXS, MS footprinting, and mutagenesis mapping of the IN C-terminal domain interaction

   PMID:22872640 PMID:23097435

   Open questions at the time

   • In vitro uncoating and intasome binding not shown to be the dominant in-cell mechanism
   • Relative contribution of direct vs indirect routes unquantified
4. 2013 High

   Resolved the principal HIV-1 mechanism as indirect—TNPO3 keeps CPSF6 nuclear, and cytoplasmic CPSF6 is the actual inhibitor—settling whether TNPO3 acts on the virus directly or through a host cargo.

   Evidence TNPO3/CPSF6 double knockdown epistasis, CPSF6 mislocalization constructs, heterologous NLS rescue, fate-of-capsid and 2-LTR circle assays across two independent studies

   PMID:23414560 PMID:23622145

   Open questions at the time

   • Does not exclude a parallel minor direct intasome route
   • Both studies from converging but limited lab sources
5. 2013 Medium

   Connected TNPO3 to human disease by identifying a frameshift mutation causing LGMD and showing the mutant fails nuclear entry, establishing a localization-based pathomechanism.

   Evidence Whole-exome sequencing with immunolocalization of mutant vs wild-type TNPO3

   PMID:23667635

   Open questions at the time

   • Localization was the sole functional readout
   • Cargo-transport consequences in muscle not measured
6. 2014 Medium

   Tested whether the direct IN-TNPO3 interaction has a separable functional role using a separation-of-function IN mutant, partially supporting a nuclear-import/integration contribution.

   Evidence IN R263A/K264A mutant analysis with qPCR import readouts, fluorescence import assay, and in vitro TNPO3 binding

   PMID:25063804

   Open questions at the time

   • Modest (2-fold) binding defect complicates causal attribution
   • Single lab, not reconciled with the CPSF6-centric model
7. 2019 Medium

   Extended the disease mechanism with a second mutation producing a C-terminally extended protein that mislocalizes away from pore complexes while partially preserving cargo transport, refining the LGMD pathomechanism.

   Evidence Gene panel sequencing, mutant construct transfection, immunofluorescence, and patient muscle protein studies

   PMID:31192305

   Open questions at the time

   • Which cargoes are selectively impaired in muscle not defined
   • Mechanistic link between mislocalization and dystrophy unresolved
8. 2019 Medium

   Identified an upstream regulatory layer in which IFN-inducible miR-128 directly downregulates TNPO3 to restrict HIV-1, integrating TNPO3 into innate antiviral control.

   Evidence miR-128 gain/loss in Jurkat and primary CD4+ T cells, luciferase TNPO3-targeting reporter, and TNPO3-independent rescue virus

   PMID:31341054

   Open questions at the time

   • Magnitude of physiological restriction in vivo unknown
   • Single lab
9. 2020 Medium

   Showed TNPO3 can import a viral cargo (RSV Gag) through a non-canonical route independent of its canonical cargo-binding domain, broadening the recognition repertoire beyond classical RS-NLS cargoes.

   Evidence Yeast genetics, direct TNPO3-Gag binding, CBD-deletion mutants, and nuclear import assay in avian cells

   PMID:32581109

   Open questions at the time

   • Structural basis of CBD-independent binding undefined
   • Generality to other cargoes untested
10. 2021 Medium

    Linked TNPO3 to muscle biology mechanistically by demonstrating SRSF1 cargo transport and dynamic redistribution during myogenesis, providing a cellular context for the dystrophy phenotype.

    Evidence Confocal, structured-illumination, and electron microscopy of TNPO3 and SRSF1 during myogenic differentiation

    PMID:33452620

    Open questions at the time

    • No loss-of-function validation of the transport requirement
    • Morphological correlation rather than functional perturbation
11. 2022 High

    Defined a non-SR cargo role: TNPO3 imports the transcription factor EBF1 via its Ig-like fold, and is required in vivo for B cell programming under antagonistic Notch signaling, broadening TNPO3 beyond splicing-factor transport.

    Evidence MS of EBF1-associated proteins, EBF1 mutant mapping (E271), conditional Tnpo3 knockout mice, RNA-seq, and chromatin binding

    PMID:36167471

    Open questions at the time

    • NLS structural basis of EBF1 recognition not solved
    • Whether Ig-like-fold recognition generalizes to other TFs unknown
12. 2023 High

    Demonstrated a splicing-specific requirement by showing Tnpo3 is needed for TCRα pre-mRNA splicing in iNKT cells, with transgenic pre-spliced cDNA rescue separating a splicing role from a developmental one.

    Evidence Tnpo3 conditional knockout mice, iNKT development analysis, and transgenic rescue with rearranged Trav11-Traj18-Trac cDNA

    PMID:37339974

    Open questions at the time

    • Direct splicing-factor cargo responsible not pinpointed
    • Generality of splicing role to other transcripts untested
13. 2025 High

    Provided the structural rules of cargo recognition, showing TNPO3 binds a non-classical RSY-NLS via tyrosine residues in a phosphorylation-independent, phosphorylation-inhibited manner, revising the canonical RS-repeat/phospho-dependent model.

    Evidence X-ray crystallography and NMR of the TNPO3-CIRBP complex with NLS mutagenesis and phosphomimetic binding assays

    PMID:40360518

    Open questions at the time

    • How phosphorylation switches differ across cargoes not generalized
    • Structural basis for RanGTP-regulated cargo release in this complex not addressed

• How TNPO3's diverse cargo-recognition modes (RS-NLS, RSY-NLS, Ig-fold TFs, CBD-independent viral cargoes) are coordinated and which selective cargo failures drive muscle pathology remain unresolved.
• No unified structural model spanning all cargo classes
• Disease-causing mutant's cargo-specific transport deficits in muscle undefined
• RanGTP-dependent cargo release mechanism not structurally resolved for most cargoes