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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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No detectable localization of hRif1 protein at telomeres in normal cells or in cells depleted of hRap1, TRF1, or TRF2 protein. (A) Western blot analysis of whole cell extracts from LOX cells treated with lentiviruses expressing hairpin siRNAs against TRF1, TRF2, or hRap1. (B) Immunofluorescence analysis of hRif1 protein in LOX melanoma cells. LOX cells were infected with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, or TRF2. 3 d after infection, cells were fixed and stained with hRif1 antibody PAB2857 (red) and TRF1 or TRF2 antibodies (green). A microscope (model Eclipse E600; Nikon) with a 100× objective and a Coolsnap fx charge-coupled device camera and software (Roper Scientific) was used to visualize the image. (C) Histogram showing the average numbers of hRif1 foci per nucleus in LOX cells after treatment with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, TRF2, or with lentiviruses expressing hTER template mutant 47A. For each treatment, hRif1 foci in ∼50 interphase cells were counted after immunofluorescence analysis and the average numbers per nucleus were presented.
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fig3: No detectable localization of hRif1 protein at telomeres in normal cells or in cells depleted of hRap1, TRF1, or TRF2 protein. (A) Western blot analysis of whole cell extracts from LOX cells treated with lentiviruses expressing hairpin siRNAs against TRF1, TRF2, or hRap1. (B) Immunofluorescence analysis of hRif1 protein in LOX melanoma cells. LOX cells were infected with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, or TRF2. 3 d after infection, cells were fixed and stained with hRif1 antibody PAB2857 (red) and TRF1 or TRF2 antibodies (green). A microscope (model Eclipse E600; Nikon) with a 100× objective and a Coolsnap fx charge-coupled device camera and software (Roper Scientific) was used to visualize the image. (C) Histogram showing the average numbers of hRif1 foci per nucleus in LOX cells after treatment with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, TRF2, or with lentiviruses expressing hTER template mutant 47A. For each treatment, hRif1 foci in ∼50 interphase cells were counted after immunofluorescence analysis and the average numbers per nucleus were presented.

Mentions: It has been demonstrated that at least a subset of S. cerevisiae Rif1p protein colocalizes with scRap1p protein at telomeres by immunofluorescence staining (Mishra and Shore, 1999; Smith et al., 2003). To examine hRif1 localization, we developed affinity-purified rabbit pAbs against peptides aa 2106–2123 and aa 2457–2472 of hRif1 (PAB2852 and PAB2857, respectively). We note that the two peptide antibodies used in the work recognize peptide sequences outside the alternatively spliced region (Fig. 1 A and Fig. S1) and thus will recognize both the longer (2472 aa) and the more abundant shorter (2446 aa) hRif1 proteins. For simplicity, in the paper we refer to hRif1 to include both the longer and the shorter hRif1 protein forms. Immunoblotting of whole cell extracts from control cells and cells depleted of hRif1 by siRNA verified that both antibodies recognized hRif1 (Fig. 1 D; not depicted). Immunostaining of tissue culture cells with each antibody showed heterogeneous nuclear staining, but no evidence of the punctate nuclear telomeric spots characteristic of TRF1, TRF2, or hRap1 staining (Fig. 3 B, top). In addition, to examine whether any fraction of hRif1 associates with known telomere binding proteins, coimmunoprecipitation experiments were performed between hRif1 and TRF1, TRF2 and hRap1 in multiple tissue culture cell lines. Unlike the mouse Rif1 which was recently reported to associate with mouse TRF2 (Adams and McLaren, 2004), no significant interactions were detected between Rif1 and TRF2, TRF1, or hRap1 (see Materials and methods; unpublished data). Together, these data suggest that hRif1 does not localize to normal human telomeres.


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

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

No detectable localization of hRif1 protein at telomeres in normal cells or in cells depleted of hRap1, TRF1, or TRF2 protein. (A) Western blot analysis of whole cell extracts from LOX cells treated with lentiviruses expressing hairpin siRNAs against TRF1, TRF2, or hRap1. (B) Immunofluorescence analysis of hRif1 protein in LOX melanoma cells. LOX cells were infected with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, or TRF2. 3 d after infection, cells were fixed and stained with hRif1 antibody PAB2857 (red) and TRF1 or TRF2 antibodies (green). A microscope (model Eclipse E600; Nikon) with a 100× objective and a Coolsnap fx charge-coupled device camera and software (Roper Scientific) was used to visualize the image. (C) Histogram showing the average numbers of hRif1 foci per nucleus in LOX cells after treatment with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, TRF2, or with lentiviruses expressing hTER template mutant 47A. For each treatment, hRif1 foci in ∼50 interphase cells were counted after immunofluorescence analysis and the average numbers per nucleus were presented.
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Related In: Results  -  Collection

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fig3: No detectable localization of hRif1 protein at telomeres in normal cells or in cells depleted of hRap1, TRF1, or TRF2 protein. (A) Western blot analysis of whole cell extracts from LOX cells treated with lentiviruses expressing hairpin siRNAs against TRF1, TRF2, or hRap1. (B) Immunofluorescence analysis of hRif1 protein in LOX melanoma cells. LOX cells were infected with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, or TRF2. 3 d after infection, cells were fixed and stained with hRif1 antibody PAB2857 (red) and TRF1 or TRF2 antibodies (green). A microscope (model Eclipse E600; Nikon) with a 100× objective and a Coolsnap fx charge-coupled device camera and software (Roper Scientific) was used to visualize the image. (C) Histogram showing the average numbers of hRif1 foci per nucleus in LOX cells after treatment with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, TRF2, or with lentiviruses expressing hTER template mutant 47A. For each treatment, hRif1 foci in ∼50 interphase cells were counted after immunofluorescence analysis and the average numbers per nucleus were presented.
Mentions: It has been demonstrated that at least a subset of S. cerevisiae Rif1p protein colocalizes with scRap1p protein at telomeres by immunofluorescence staining (Mishra and Shore, 1999; Smith et al., 2003). To examine hRif1 localization, we developed affinity-purified rabbit pAbs against peptides aa 2106–2123 and aa 2457–2472 of hRif1 (PAB2852 and PAB2857, respectively). We note that the two peptide antibodies used in the work recognize peptide sequences outside the alternatively spliced region (Fig. 1 A and Fig. S1) and thus will recognize both the longer (2472 aa) and the more abundant shorter (2446 aa) hRif1 proteins. For simplicity, in the paper we refer to hRif1 to include both the longer and the shorter hRif1 protein forms. Immunoblotting of whole cell extracts from control cells and cells depleted of hRif1 by siRNA verified that both antibodies recognized hRif1 (Fig. 1 D; not depicted). Immunostaining of tissue culture cells with each antibody showed heterogeneous nuclear staining, but no evidence of the punctate nuclear telomeric spots characteristic of TRF1, TRF2, or hRap1 staining (Fig. 3 B, top). In addition, to examine whether any fraction of hRif1 associates with known telomere binding proteins, coimmunoprecipitation experiments were performed between hRif1 and TRF1, TRF2 and hRap1 in multiple tissue culture cell lines. Unlike the mouse Rif1 which was recently reported to associate with mouse TRF2 (Adams and McLaren, 2004), no significant interactions were detected between Rif1 and TRF2, TRF1, or hRap1 (see Materials and methods; unpublished data). Together, these data suggest that hRif1 does not localize to normal human telomeres.

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