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Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules.

Xu L, Blackburn EH - J. Cell Biol. (2004)

Bottom Line: The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes.In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear.These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

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hRif1 localizes on uncapped telomeres without a requirement for wild-type p53. HCT116 cell lines with the p53 locus wild type or disrupted, but which were otherwise isogenic, were infected with lentiviruses expressing 47A-hTer. 5 d after infection, cells were fixed and stained with 53BP1 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described for Fig. 4. Bars, 10 μm.
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fig5: hRif1 localizes on uncapped telomeres without a requirement for wild-type p53. HCT116 cell lines with the p53 locus wild type or disrupted, but which were otherwise isogenic, were infected with lentiviruses expressing 47A-hTer. 5 d after infection, cells were fixed and stained with 53BP1 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described for Fig. 4. Bars, 10 μm.

Mentions: It has been demonstrated that telomeres deficient in TRF2 binding induce a p53-dependent apoptotic response (Karlseder et al., 1999). To explore the role of p53 in the response of hRif1 to mutant-sequence telomeres, we expressed telomerase RNA template mutant 47A in two human colon cancer cell lines that are isogenic except for their p53 status, HCT116 (p53WT) and HCT116 (p53−/−) (Bunz et al., 1998). Indirect immunofluorescence staining demonstrated that hRif1 translocated to discrete DNA damage foci as indicated by 53BP1 accumulation upon expression of template mutant 47A in both cell lines with similar efficiency (Fig. 5). This result indicated that hRif1 can respond to damaged telomeres without a requirement for p53.


Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules.

Xu L, Blackburn EH - J. Cell Biol. (2004)

hRif1 localizes on uncapped telomeres without a requirement for wild-type p53. HCT116 cell lines with the p53 locus wild type or disrupted, but which were otherwise isogenic, were infected with lentiviruses expressing 47A-hTer. 5 d after infection, cells were fixed and stained with 53BP1 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described for Fig. 4. Bars, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172464&req=5

fig5: hRif1 localizes on uncapped telomeres without a requirement for wild-type p53. HCT116 cell lines with the p53 locus wild type or disrupted, but which were otherwise isogenic, were infected with lentiviruses expressing 47A-hTer. 5 d after infection, cells were fixed and stained with 53BP1 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described for Fig. 4. Bars, 10 μm.
Mentions: It has been demonstrated that telomeres deficient in TRF2 binding induce a p53-dependent apoptotic response (Karlseder et al., 1999). To explore the role of p53 in the response of hRif1 to mutant-sequence telomeres, we expressed telomerase RNA template mutant 47A in two human colon cancer cell lines that are isogenic except for their p53 status, HCT116 (p53WT) and HCT116 (p53−/−) (Bunz et al., 1998). Indirect immunofluorescence staining demonstrated that hRif1 translocated to discrete DNA damage foci as indicated by 53BP1 accumulation upon expression of template mutant 47A in both cell lines with similar efficiency (Fig. 5). This result indicated that hRif1 can respond to damaged telomeres without a requirement for p53.

Bottom Line: The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes.In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear.These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

Show MeSH
Related in: MedlinePlus