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  • However recent studies revealed that the

    2020-09-16

    However, recent studies revealed that the function of Crm1 might not only limit to nucleocytoplasmic transport, but also be involved in regulation of centrosome duplication. As the major microtubule organizing centers in animal cells, centrosomes play a significant role in cell cycle, spindle formation and cytokinesis . Forgues and colleagues reported that Crm1 is related with Hepatitis B virus X protein-induced centrosomes multireplication and multipolar spindle formation . Years later, nucleophosmin was demonstrated to regulate the centrosome duplication by associating with Crm1 . The NES defective mutation of nucleophosmin or LMB treatment dissociated nucleophosmin from centrosomes in early G1 phase and caused premature centrosome duplication . Then, many other factors, such as p53 and Brca2, were reported to be involved in centrosome duplication under the regulation of Crm1 . Moreover, a recent study revealed that Crm1 might also regulate the microtubules nucleation and spindle formation by associating with the centrosomal structural protein Spc72 in WM-1119 yeast . Crm1 regulates these factors by interacting with their NES and targeting them spatially and temporally to centrosomes. It is likely that Crm1 supplies a docking site for these factors at centrosomes and regulates their localization and function . But it is still unknown the mechanism for Crm1 to target to centrosomes. In this study, through observing the subcellular localization of truncated mutants of Crm1, we found that Crm1 might interact with centrosomal RanGTP through its CRIME domain. Moreover, overexpression of CRIME domain or deficiency of Crm1 in cells by RNAi would lead to loss of pericentrin and γ-tubulin from centrosomes. Pericentrin is a conserved centrosomal structural protein, which anchors γ-tubulin to centrosomes and play a key role in microtubule nucleation and mitotic spindles organization , , . Since pericentrin was reported to be sensitive to LMB , we propose that Crm1 might be targeted to centrosomes by centrosomal RanGTP and might affect the centrosomal localization and function of pericentrin by interacting with pericentrin. Materials and methods Plasmids. XCrm1 cDNA was amplified from the Xenopus XTC cells cDNA library by PCR and ligated into pGEX4T-1 (Amersham) and pEGFPC1 (Clontech). The truncated mutant Crm1 (809–1071) was amplified from the full length Crm1 cDNA by PCR and cloned into the pET32a (Novagen). The truncated mutants (residues 1–417, 417–601, 601–1071, 1–112, 112–417, 1–310, 310–417 and 112–310) of Crm1 were amplified from the full length Crm1 cDNA by PCR and cloned into the pEGFPC1 vector. RNA interference. The DNA fragment for the expression of shRNA targeting the positions 287–305 of human Crm1 was generated by annealing the sense oligonucleotide 5′-GATCCCCGGAACCAGTGCGAAGGAATTTCAAGAGAATTCCTTCGCACTGGTTCCTTTTTA-3′ and the anti-sense oligonucleotide 3′-GGGCCTTGGTCACGCTTCCTTAAAG TTCTCTTAAGGAAGCGTGACCAAGGAAAAATTCGA-5′ and ligated to pSUPER plasmid. The control pSUPER vector was constructed using a 19-nt sequence (5′-GCGCGCTTTGTAGGATTCG-3′) with no significant homology to any mammalian gene sequence. Expression of recombinant protein. GST-Crm1 protein was purified using Glutathione Sepharose-4B (Pharmacia) affinity resin. His-tagged Crm1 (805–1071) truncated protein was purified using TALON Metal affinity resin (Clontech). Cell culture and cell transfection. HeLa and NIH3T3 cells were cultured in DMEM (Invitrogen) with 10% fetal bovine serum (HyClone), 100U/ml penicillin and 100μg/ml streptomycin at 37°C, 5% CO2. XTC cells were cultured in DMEM with 10% fetal bovine serum, 25% ddH2O, 100U/ml penicillin and 100μg/ml streptomycin at 23°C. Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the protocol provided by the manufacturer. To deplete the endogenous Crm1, RFP-H2B was co-transfected with pSuper-Crm1 at a 1:20 ratio to indicate the pSuper-Crm1 transfected cells.