Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences

Abstract

Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluoresceinconjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time-and energydependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin ␣, importin ␤, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import.The nuclear envelope presents an effective barrier between the nuclear and cytoplasmic compartments of the cell. Although it is impermeant to large non-nuclear molecules, a multitude of macromolecules must enter and exit the nucleus across this envelope every second in order for the cell to live. All macromolecular exchange between the nucleus and the cytoplasm studied to date occurs through the nuclear pore complex (NPC), 1 is signal-dependent, and utilizes a series of receptor proteins (for a review see Ref. 1). In the case of proteins destined for the nucleus, the nuclear localization signal (NLS) interacts with one of a growing number of importin family members to target the complex to the NPC. In the classical case of NLS-containing proteins, the protein binds to importin ␣, the NLS "receptor," which in turn interacts with importin ␤. Once at the NPC, the complex interacts with the small GTP-binding protein RAN in its GDP-bound state and its accessory factor NTF2 while being translocated across the NPC. After translocation into the nucleus, the complex disassembles because of the conversion of RAN-GDP to RAN-GTP by exchange or replacement and the importins return to the cytoplasm (2). Similar scenarios of receptor proteins interacting with signals to mediate translocation across the nuclear envelope also occur for the nuclear export of proteins (e.g. exportin and the nuclear export signal) and viral mRNAs (Crm1p, HIV Rev, and the Rev response element), and the nuclear import of small nuclear RNAs that contain both protein-encoded NLSs and the trimethylguanosine cap as import signals (1, 3).We have recently shown that the nuclear import of plasmid DNA (pDNA) also u...

Transfection of large plasmids in primary human myoblasts

“…pEPI-EGFP, pMDysE and pDysE contain the SV40 sequences determined to permit optimal DNA entry through nuclear pores. 52 In conclusion, branched 2 kDa PEI was less toxic in PEI adenofection than branched 25 kDa PEI or linear 22 kDa PEI. The adenovirus was necessary to transfect plasmids of 12 kb or more in primary human myoblasts.…”

Sequence Requirements for Plasmid Nuclear Import

“…Thus, the complex is recognized through the NLS by the protein import machinery and is thereby targeted to the nucleus (2). Indeed, in experiments using digitonin-permeabilized HeLa cells, we have shown recently that nuclear import of plasmids can occur only when both nuclear extract and recombinant importinα, importinβ, and RAN are provided; neither nuclear extract alone nor the purified NLS-import machinery proteins alone can suffice (17). Of the 372 bp in the SV40 origin/promoter region, the enhancer contains the most number of consensus binding sites for such proteins.…”

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

“…We, and others, have subsequently shown that nuclear import of plasmid DNA is sequence dependent, requires karyopherins (importins) and the small GTPase RAN, and occurs through the nuclear pore complex (NPC). [14][15][16][17][18] In our model of plasmid nuclear import, we propose that transcription factors present in the cytoplasm bind to the DTS and coat the plasmid with nuclear localization sequences (NLSs) that utilize importins to cross the NPC, which regulates the nucleocytoplasmic shuttling of macromolecules during interphase. [19][20][21] We have also developed a tissue-specific DTS, utilizing the smooth muscle g-actin (SMGA) promoter, that mediates nuclear import of pDNA specifically in smooth muscle cells (SMCs).…”