Identification of protein cofactors necessary for sequence-specific plasmid DNA nuclear import

Abstract

Although transfections are routinely used in the laboratory, the mechanism(s) by which exogenous DNA is transported into the nucleus is poorly understood. By improving our understanding of how vectors circumvent the numerous cellular barriers to gene transfer, more efficient gene delivery methods can be devised. We have begun to design plasmid constructs that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing levels of expression. We have shown that inclusion of specific DNA sequences in plasmid constructs mediates nuclear import both in vitro and in vivo. Here, we use plasmid affinity chromatography, mass spectrometry (MS), and live-cell pulldowns of transfected plasmid constructs to identify protein cofactors that interact in a sequence-specific manner with these DNA nuclear targeting sequences (DTSs). Importin beta(1), importin 7, and the small guanosine triphosphatase Ran all demonstrate DTS-specific interaction in both MS and pull-down assays, consistent with our model of plasmid nuclear import. In addition, knockdown of importin beta(1) with small interfering RNA (siRNA) abrogates plasmid nuclear import, indicating that it is a necessary cofactor. Our discovery that specific karyopherins mediate plasmid nuclear import can be used to design more effective vectors for gene delivery.

Figure 1

Purification of smooth muscle cell extracts that interact with the SMGA DTS. Sepharose beads were linked to peptide nucleic acid (PNA) clamps and hybridized to plasmids that contain or lack the SMGA DTS (pTOPO-DTS and pTOPO, respectively). Affinity chromatography was performed using whole-cell smooth muscle extracts to purify factors that interact with the plasmids. (a) A chromatogram showing the milli-absorbance units absorbance spectra of eluted protein and quantification of total eluted protein. A two-step salt elution (0.4 mol/l and 1.0 mol/l) was used. (b) Diagram of the Sepharose-PNA-pDNA phase. Sepharose beads are covalently linked to the PNA by an isourea bond. The amino-terminal half of the PNA hybridizes to the plasmid target sequence by Watson-Crick (WC) base pairs and the carboxy-terminal half uses Hoogstein base pairs to form a triplex. 8-amino-3,6-dioxaoctanoic acid linkers connect the two halves of the PNA (abbreviated a “O”). The displaced “D-loop” is shown bulging away from the complementary DNA strand. (c) Immunoblots of eluted protein showing that Ran and importin β₁ (but not importin α₁) are purified from affinity columns in a DTS-specific manner. DTS, DNA nuclear targeting sequence; SMGA, smooth muscle gamma actin.

Figure 2

Classification of proteins purified from plasmid DNA affinity columns. The functional classes of proteins identified by mass spectrometry analysis of column eluates from (a) SMGA DTS columns and (b) No DTS columns are shown as the total number of identified proteins. Classifications are based on data acquired through National Center for Biotechnology Information.

Figure 3

Precipitation of the protein–pDNA import complex in living cells. (a) Human smooth muscle cells were electroporated with plasmid DNA (pDNA) that was hybridized to a biotinylated peptide nucleic acid clamp. At 60 and 240 minutes post-transfection, cells were treated with formaldehyde to cross-link proteins bound to the pDNA, lysed and precipitated using streptavidin-agarose. Unbound fractions (Sup) and precipitated fractions (Pel) were probed for putative members of the import complex using western blots. (b) To verify equal levels of transfection efficiency, crude lysates from transfected cells were Slot blotted onto membranes, UV cross-linked and probed for biotinylated pDNA using horseradish peroxidase–streptavidin. DTS, DNA nuclear targeting sequence; SMGA, smooth muscle gamma actin; SV40, simian virus 40.

Figure 4

Importin β₁ is required for DTS-mediated plasmid nuclear import. (a) For microinjection experiments, Cy3-labeled plasmid DNA (pDNA) (0.5 mg/ml) and fluorescein-labeled (Fl) M9 NLS peptide (0.5 mg/ml) were microinjected into the cytoplasm of adherent cells and their localization was determined 8 hours later using fluorescence microscopy. Representative examples of import and no import are shown. (b) Immunoblots of human smooth muscle cells (hSMCs) transfected with nontargeting (NT) small interfering RNA (siRNA) or siRNA targeting importin β₁ and importin 7 reveal successful knockdown of each factor at 48 hours. (c) Quantification of levels of nuclear import of plasmid constructs cytoplasmically microinjected into hSMCs that were treated with siRNA for 48 hours. At least 100 cells were injected for each condition and the experiment was repeated three times (mean % nuclear import ± SD). *P < 0.001 versus respective NT control. DAPI, 4,6-diamidino-2-phenylindole; DTS, DNA nuclear targeting sequence; NLS, nuclear localization signal; SMGA, smooth muscle gamma actin; SV40, simian virus 40.