The Renpenning syndrome-associated protein PQBP1 facilitates the nuclear import of splicing factor TXNL4A through the karyopherin β2 receptor

Abstract

Renpenning syndrome belongs to a group of X-linked intellectual disability disorders. The Renpenning syndrome-associated protein PQBP1 (polyglutamine-binding protein 1) is intrinsically disordered, associates with several splicing factors, and is involved in pre-mRNA splicing. PQBP1 uses its C-terminal YxxPxxVL motif for binding to the splicing factor TXNL4A (thioredoxin like 4A), but the biological function of this interaction has yet to be elucidated. In this study, using recombinant protein expression, in vitro binding assays, and immunofluorescence microscopy in HeLa cells, we found that a recently reported X-linked intellectual disability-associated missense mutation, resulting in the PQBP1-P244L variant, disrupts the interaction with TXNL4A. We further show that this interaction is critical for the subcellular location of TXNL4A. In combination with other PQBP1 variants lacking a functional nuclear localization signal required for recognition by the nuclear import receptor karyopherin β2, we demonstrate that PQBP1 facilitates the nuclear import of TXNL4A via a piggyback mechanism. These findings expand our understanding of the molecular basis of the PQBP1-TXNL4A interaction and of the etiology and pathogenesis of Renpenning syndrome and related disorders.

Keywords: PQBP1; Renpenning syndrome; TXNL4A; X-linked intellectual disability; autism; neurodevelopment; nuclear transport; protein complex; protein import; protein-protein interaction; splicing factor.

Figure 1.

The C-terminal mutations of PQBP1 disrupt its interaction with TXNL4A. A, schematic demonstration of protein domain structures of PQBP1–WT and mutants. The mutated regions are highlighted with red. Y65C, the missense mutant in the WW domain; Δ461–462, frameshift mutant c.461–462del, p.E154Afs*12; Δ575–576, frameshift mutant c.575–576del, p.K192Sfs*7; P244L, the missense mutation in C-terminal domain; RPY, tetra-point mutant (¹⁸⁰RR¹⁸¹ and ¹⁸⁶PY¹⁸⁷ to alanines) in the nuclear localization signal. B, in vitro binding assays show the interactions of GST–PQBP1–WT and mutants with TXNL4A. Immobilized GST–PQBP1–WT and mutants were incubated with purified recombinant TXNL4A. Representative results from three independent experiments. C, densitometric analysis of B. The relative density of TXNL4A band against GST–PQBP1 band in each reaction was normalized to that in the reaction with GST–PQBP1–WT (100%). The data show as means ± S.D. from three independent experiments. ****, p < 0.0001. MW, molecular mass.

Figure 2.

The C-terminal mutations of PQBP1 disrupt its interaction with TXNL4A. A, structure mutagenesis analysis on the complex of PQBP1–CT43 and TXNL4A. The structure of PQBP1–WT and TXNL4A was adopted from Protein Data Bank structure 4BWQ. Green cartoon, PQBP1; gray surface, TXNL4A; yellow surface, the three residues Lys⁸⁸, His⁸⁹, and Met⁹¹ in TXNL4A that form the small cavity accommodating Pro²⁴⁴ in PQBP1. Mutagenesis analysis was done with PyMOL, and the side chains of mutated residues are shown as stick model and highlighted with red. B, in vitro binding assays show the interactions of GST–PQBP1–WT and P244 site mutants with TXNL4A. Immobilized GST–PQBP1–WT and mutants were incubated with purified recombinant TXNL4A. Representative results from three independent experiments. C, densitometric analysis of B. The relative density of TXNL4A band against GST–PQBP1 band in each reaction was normalized to that in the reaction with GST–PQBP1–WT (100%). The data are shown as means ± S.D. from three independent experiments. **, p < 0.01; ***, p < 0.001. MW, molecular mass.

Figure 3.

PQBP1 binds to the nuclear import receptor Kapβ2 through its PY-NLS. A, the sequences of PQBP1–WT and RPY mutants at PY-NLS. The three-epitope structure of PY-NLS consensus sequence is shown at the top. The mutated residues are highlighted with red. B, in vitro binding assays show the interactions of GST–PQBP1–WT and mutants with Kapβ2. Immobilized GST–PQBP1–WT and mutants were incubated with purified recombinant Kapβ2. Representative results from three independent experiments are shown here. C, densitometric analysis of B. The relative density of Kapβ2 band against GST–PQBP1 band in each reaction was normalized to that in the reaction with GST–PQBP1–WT (100%). The data are shown as means ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; ****, p < 0.0001. MW, molecular mass.

Figure 4.

PQBP1 binds Kapβ2 and TXNL4A simultaneously. A, in vitro binding assays show the interactions of GST–PQBP1–WT with Kapβ2 and TXNL4A. Immobilized GST–PQBP1–WT was incubated with purified recombinant TXNL4A alone, Kapβ2 alone, or Kapβ2 with TXNL4A. Shown are representative results from three independent experiments. B, Western blots show the interactions of MBP–TXNL4A with PQBP1 and Kapβ2. Immobilized MBP–TXNL4A was incubated with purified recombinant Kapβ2 in the presence or absence of PQBP1–WT or mutants. Representative results from three independent experiments. MW, molecular mass.

Figure 5.

PQBP1 facilitates the nuclear import of TXNL4A. A, immunostaining shows the subcellular localizations of transfected PQBP1 and TXNL4A. HeLa cells were transfected with pFLAG–CMV2–PQBP1–WT or mutants in the presence of pEGFP–C1–TXNL4A. Scale bar, 20 μm. B, quantification of the nuclear/cytoplasmic fluorescent intensities of EGFP-TXNL4A signals. The data show the means ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01. C, the model of PQBP1-facilitated nuclear import of TXNL4A via the Kapβ2-mediated pathway. DAPI, 4′,6′-diamino-2-phenylindole.