Long interspersed nuclear element-1 (LINE-1) is the only currently active retrotransposon in humans and encodes the proteins ORF1p and ORF2p. Retrotransposition proceeds via a ‘copy-and-paste’ mechanism. This means that LINE-1 is first transcribed into mRNA within the nucleus. Following export to the cytoplasm, it is translated into proteins that form a ribonucleoprotein complex with their own mRNA and are transported back into the nucleus. There, retrotransposition occurs via target-primed reverse transcription. Since this process is known to take place in non-dividing cells—such as neurons—ORF1p, ORF2p, and LINE-1 mRNA must be able to enter the nucleus. The aim of this master's thesis was to elucidate the mechanism of ORF1p nuclear import.
First, we expressed and isolated the following recombinant proteins in E. coli: ORF1p, the nuclear localization signal of the simian virus 40 large T antigen, karyopherin alpha 6 (KPNA6), karyopherin beta 1 (KPNB1), and Ran. All constructs carried an N-terminal glutathione S-transferase fusion tag. Nuclear transport was investigated using a nuclear import assay, in which the plasma membrane of HeLa cells was selectively permeabilized. After removing the cytoplasm, we added import mixtures to the nuclei containing the recombinant ORF1p protein, nuclear import factors, and an ATP-regenerating system. We first used reticulocyte lysate as a nuclear import factor and demonstrated that ORF1p actively enters the nucleus and that this transport depends on the presence of karyopherins. We then replaced the reticulocyte lysate with individual karyopherins and Ran. The results of these experiments suggest that the nuclear import of ORF1p likely involves the KPNA6/KPNB1 complex.
These results contribute to an understanding of the mechanism of ORF1p nuclear transport and, consequently, the regulation of the LINE-1 retrotransposition process.
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