TNPO3

TNPO3 (Transportin-3/TRN-SR2) is a nuclear import receptor of the importin-β superfamily that transports serine/arginine-rich (SR) splicing factors and other cargoes into the nucleus, thereby coupling nuclear import to RNA splicing, lymphocyte differentiation, and myogenesis. Its cargo-binding domain recognizes RS/SR-repeat nuclear localization signals as well as non-classical RSY-type NLS motifs in which tyrosine residues are critical for binding, with serine and tyrosine phosphorylation acting as negative regulators of cargo recognition (PMID:40360518). TNPO3 is co-opted during HIV-1 infection: its primary contribution to viral replication is maintaining the nuclear localization of the capsid-binding factor CPSF6, whose cytoplasmic accumulation upon TNPO3 depletion abnormally stabilizes the HIV-1 core and blocks infection at the nuclear import/integration step (PMID:23414560, PMID:23622145); TNPO3 also directly stimulates HIV-1 core uncoating in vitro in a RanGTP-sensitive manner and binds the HIV-1 intasome through the integrase C-terminal domain (PMID:23097435, PMID:22872640). In normal physiology, conditional Tnpo3 knockout in mice blocks early B cell differentiation through interaction with the transcription factor EBF1, and Tnpo3 deficiency impairs iNKT cell development by disrupting splicing of the semi-invariant TCRα pre-mRNA, a defect rescued by a pre-spliced cDNA transgene (PMID:36167471, PMID:37339974); heterozygous frameshift mutations in TNPO3 cause limb-girdle muscular dystrophy type D2 (LGMD D2) (PMID:23667635, PMID:31192305).

1. 2009 High

   Establishing that HIV-1 capsid (CA), not integrase, is the dominant viral determinant of TNPO3 dependency resolved the initial confusion over which viral component TNPO3 functionally engages during infection.

   Evidence MLV/HIV-1 chimeric virus infectivity assays in TNPO3-knockdown cells with surface plasmon resonance binding comparisons

   PMID:19846519

   Open questions at the time

   • How CA dictates TNPO3 dependency if TNPO3 does not directly bind assembled cores remained unresolved
   • No structural basis for the CA-TNPO3 functional link
2. 2011 High

   Mapping the TNPO3-dependent step in HIV-1 replication to a point after nuclear entry (post-2-LTR circle formation but before integration) revealed that TNPO3 acts beyond classical nuclear import, and a systematic panel of 27 CA mutants confirmed CA as the genetic determinant.

   Evidence TNPO3 knockdown with qPCR quantification of viral cDNA, 2-LTR circles, and proviral DNA across 27 CA mutants in multiple cell types

   PMID:22145813 PMID:22176773

   Open questions at the time

   • Whether the nuclear-stage effect reflects a direct TNPO3 activity or an indirect consequence via a host co-factor was unknown
3. 2012 High

   Biophysical and biochemical characterization showed TNPO3 directly binds the HIV-1 intasome (not naked DNA or capsid cores) through the IN C-terminal domain and the TNPO3 cargo-binding domain, while a separate in vitro reconstitution demonstrated TNPO3 stimulates HIV-1 core uncoating in a RanGTP-sensitive manner, establishing two distinct biochemical activities relevant to HIV-1.

   Evidence Purified recombinant protein binding assays, mass spectrometry footprinting, mutagenesis (intasome binding); in vitro core uncoating assay with RanGTP and CypA (uncoating activity)

   PMID:22872640 PMID:23097435

   Open questions at the time

   • Whether in vitro uncoating activity reflects the physiologically dominant mechanism was unclear
   • The relative contributions of intasome binding versus uncoating stimulation to infectivity were not deconvolved
4. 2013 High

   The discovery that TNPO3's principal role in HIV-1 infection is maintaining nuclear localization of the CA-binding factor CPSF6 — with cytoplasmic CPSF6 being the actual restriction factor that stabilizes cores and blocks nuclear import — unified the CA-dependency and post-nuclear-entry observations into a coherent indirect mechanism.

   Evidence TNPO3/CPSF6 double knockdown rescue; heterologous NLS fusion rescue of CPSF6; fate-of-capsid assays; 2-LTR circle qPCR

   PMID:23414560 PMID:23622145

   Open questions at the time

   • Whether TNPO3 has any CPSF6-independent contribution to HIV-1 replication remained debated
   • How CPSF6 nuclear import by TNPO3 is regulated was not resolved
5. 2013 Medium

   Identification of a heterozygous frameshift mutation in TNPO3 as the cause of LGMD D2 established TNPO3 as a disease gene for muscular dystrophy and showed that mutant TNPO3 mislocalizes to perinuclear regions rather than entering the nucleus.

   Evidence Whole-exome sequencing of LGMD1F families; Sanger validation; subcellular localization of mutant TNPO3 by immunofluorescence

   PMID:23667635

   Open questions at the time

   • The downstream molecular pathway from impaired nuclear import to muscle degeneration was not defined
   • Whether dominant-negative effects or haploinsufficiency underlies pathology was unclear
6. 2014 Medium

   The IN R263A/K264A double mutant that selectively disrupts TNPO3 binding while preserving reverse transcription resulted in impaired nuclear import and integration, supporting a direct functional role for the IN-TNPO3 interaction in HIV-1 nuclear entry.

   Evidence Site-directed IN mutagenesis; TNPO3 binding assay; qPCR for 2-LTR circles and integration; eGFP-IN imaging

   PMID:25063804

   Open questions at the time

   • Whether this nuclear import defect is separable from the CPSF6-mediated indirect mechanism was not tested
   • Single-lab finding without independent replication
7. 2019 Medium

   A second LGMD D2-causing TNPO3 frameshift mutation confirmed the disease locus and showed that mutant protein accumulates at subsarcolemmal/perinuclear sites and fails to localize to annulate lamellae, while at least some SR-protein cargoes retain normal nuclear localization.

   Evidence Genetic sequencing of independent family; immunofluorescence of patient muscle and transfected cells

   PMID:31192305

   Open questions at the time

   • Which specific cargo(es) are critically mis-imported in LGMD D2 muscle remains unknown
   • No functional rescue experiment in patient cells
8. 2020 Medium

   Demonstration that TNPO3 binds RSV Gag and mediates its nuclear entry through a mechanism independent of the canonical cargo-binding domain broadened the repertoire of TNPO3 import mechanisms beyond SR-domain recognition.

   Evidence TNPO3 CBD deletion mutants; co-immunoprecipitation; nuclear import assays in avian cells

   PMID:32581109

   Open questions at the time

   • The binding site on TNPO3 for Gag was not mapped at residue resolution
   • Whether this non-CBD mechanism extends to other cargo proteins is unknown
9. 2022 High

   Conditional knockout of Tnpo3 in the B lineage revealed that TNPO3 is required for early B cell differentiation by interacting with EBF1 through its immunoglobulin-like domain (E271), with loss of Tnpo3 causing downregulation of B lineage genes and aberrant upregulation of T/NK genes.

   Evidence EBF1 interactome mass spectrometry; co-immunoprecipitation; EBF1 E271A point mutant retroviral complementation in Ebf1−/− progenitors; Tnpo3 conditional KO mice; RNA-seq

   PMID:36167471

   Open questions at the time

   • Whether TNPO3 acts by importing EBF1 into the nucleus or by an import-independent mechanism was not fully resolved
   • The splicing targets relevant to the B cell differentiation block were not identified
10. 2023 High

    Tnpo3 was shown to be essential for iNKT cell development specifically by enabling correct splicing of the Trav11-Traj18-Trac pre-mRNA, as a pre-spliced cDNA transgene fully rescued the iNKT cell defect, definitively positioning TNPO3's physiological function at the nuclear import-to-splicing axis.

    Evidence Tnpo3-deficient mouse model; iNKT cell flow cytometry; pre-spliced TCRα cDNA transgenic rescue; pre-mRNA splicing analysis

    PMID:37339974

    Open questions at the time

    • Which SR-protein cargo(es) are rate-limiting for TCRα splicing downstream of TNPO3 import is unknown
    • Whether analogous splicing defects occur in other Tnpo3-dependent cell lineages was not tested
11. 2025 High

    The crystal structure of TNPO3 bound to CIRBP revealed a non-classical RSY-NLS recognition mode in which tyrosine residues (not serine phosphorylation) drive binding, with phosphorylation of serine and tyrosine residues inhibiting the interaction, establishing a phosphorylation-regulated import mechanism distinct from canonical RS-domain recognition.

    Evidence X-ray crystallography of TNPO3–CIRBP complex; tyrosine mutagenesis; phosphopeptide binding assays

    PMID:40360518

    Open questions at the time

    • Whether the RSY-NLS mode applies to other TNPO3 cargoes beyond CIRBP is untested
    • Structural basis for how phosphorylation blocks binding at atomic resolution is incomplete

• Major unresolved questions include which specific cargo(es) are critically mis-imported in LGMD D2 muscle, whether TNPO3 has CPSF6-independent contributions to HIV-1 replication in vivo, and the full repertoire of physiological cargoes recognized via the RSY-NLS versus classical RS-domain mechanisms.
• No systematic cargo profiling in TNPO3-deficient cells has been reported
• The pathogenic mechanism of LGMD D2 at the molecular cargo level is undefined
• No structural basis for the TNPO3–CPSF6 interaction has been determined