Nuclear Import Pathway of the Telomere Elongation Supressor TRF1: Inhibition by Importin α




Forwood, Jade
Jans, David A

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American Chemical Society


Telomere repeat factor 1 (TRF1) regulates the steady-state length of chromosomes, whereby its overexpression results in telomere shortening while dominant negative TRF1 mutations can lead to telomere elongation, which is linked to cell immortalization/transformation. Although present in the nucleus at mammalian chromosomal ends during interphase and mitosis, nothing is known of the mechanism by which TRF1 enters the nucleus or how its nuclear levels may be regulated and the relevance of this, in turn, to telomere length and cell immortalization. Here we examine the nuclear import mechanism of TRF by expressing and purifying a recombinant TRF1-GFP (green fluorescent protein) fusion protein that is functional in terms of being able to bind telomeric DNA specifically as shown using a novel, quantitative double-label gel mobility shift assay. We quantitate the ability of TRF1-GFP to accumulate in the nucleus using real time confocal laser scanning microscopy, showing that the nuclear import pathway of TRF1 is mediated by importin (Imp) β1 and Ran. Impβ is shown to bind directly to TRF1 with nanomolar affinity using native gel electrophoretic and fluorescence polarization (FP) approaches; FP experiments also demonstrate that Impβ residues 1-380 are responsible for TRF1 binding. Intriguingly, when dimerized to Impβ, Impα was found to inhibit Impβ-mediated nuclear accumulation, although not affecting Impβ binding to TRF1. The study represents the first elucidation of the nuclear transport mechanism of TRF1; that its nuclear import is mediated directly by Impβ but inhibited by Impα may represent a novel regulatory mechanism, with potential relevance to oncogenesis.



Keywords: Oncogenesis; Bioassay; Cells; Chromosomes; Dimerization; DNA; Electrophoresis; Fluorescence; Polarization; Proteins; Scanning electron microscopy; Biochemistry; green fluorescent protein; karyopherin; karyopherin alpha; karyopherin beta; regulator protein





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