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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 overexpression in S. cerevisiae perturbs telomere length maintenance dependent on the presence of the scRif1 gene. (A) Southern blot of telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Genomic DNA was isolated from yeast cells, digested with XhoI, and hybridized with a telomeric probe. (B) Histogram of bulk telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Length of peak telomeric restriction fragments was quantified with ImageQuant from five independent sporulation experiments and is shown as mean ± SD.
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fig6: hRif1 overexpression in S. cerevisiae perturbs telomere length maintenance dependent on the presence of the scRif1 gene. (A) Southern blot of telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Genomic DNA was isolated from yeast cells, digested with XhoI, and hybridized with a telomeric probe. (B) Histogram of bulk telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Length of peak telomeric restriction fragments was quantified with ImageQuant from five independent sporulation experiments and is shown as mean ± SD.

Mentions: Having shown that human Rif1 can localize to telomeres when they are made aberrant, we further investigated any potential telomeric role of hRif1 by testing whether the protein retains any evolutionarily conserved functional property across species. Because S. cerevisiae Rif1p and Rif2p negatively regulate telomere length (Wotton and Shore, 1997), we examined the effect of hRif1 overexpression on telomeric tract length in S. cerevisiae in wild-type yeast cells and in cells deleted for the scRif1 gene, scRif2 gene, or both. The hRif1 full-length cDNA was cloned into a yeast 2μ-plasmid under the control of the ADH1 promoter and transformed into a RIF1/rif1, RIF2/rif2 heterozygous S. cerevisiae strain. The empty vector was used as a control. The resulting strains were sporulated and spores with desired genotypes overexpressing hRif1 or vector alone were selected and passaged on plates consecutively for five streaks (∼125 population doublings) to allow telomere length to reach equilibrium. We then compared the effects of human Rif1 overexpression in the wild-type, Δrif1, Δrif2 and Δrif1Δrif2 mutant yeast cells. Data from five independent sporulation experiments showed that overexpression of hRif1 in wild-type and Δrif2 strains led to significant telomere elongation; mean telomere length averaged 330 and 910 bp longer, respectively. In contrast, overexpression of hRif1 in Δrif1 or Δrif1Δrif2 strains did not induce significant telomere length changes compared with the vector control (changes ≤120 bp; Fig. 6, A and B; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1). The finding that hRif1 overexpression perturbs telomere length maintenance, in a manner specifically dependent on the presence of the S. cerevisiae Rif1 gene, suggests that hRif1 and scRif1p proteins share at least some conserved functional interactions that can directly or indirectly affect telomeres.


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

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

hRif1 overexpression in S. cerevisiae perturbs telomere length maintenance dependent on the presence of the scRif1 gene. (A) Southern blot of telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Genomic DNA was isolated from yeast cells, digested with XhoI, and hybridized with a telomeric probe. (B) Histogram of bulk telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Length of peak telomeric restriction fragments was quantified with ImageQuant from five independent sporulation experiments and is shown as mean ± SD.
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Related In: Results  -  Collection

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

fig6: hRif1 overexpression in S. cerevisiae perturbs telomere length maintenance dependent on the presence of the scRif1 gene. (A) Southern blot of telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Genomic DNA was isolated from yeast cells, digested with XhoI, and hybridized with a telomeric probe. (B) Histogram of bulk telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Length of peak telomeric restriction fragments was quantified with ImageQuant from five independent sporulation experiments and is shown as mean ± SD.
Mentions: Having shown that human Rif1 can localize to telomeres when they are made aberrant, we further investigated any potential telomeric role of hRif1 by testing whether the protein retains any evolutionarily conserved functional property across species. Because S. cerevisiae Rif1p and Rif2p negatively regulate telomere length (Wotton and Shore, 1997), we examined the effect of hRif1 overexpression on telomeric tract length in S. cerevisiae in wild-type yeast cells and in cells deleted for the scRif1 gene, scRif2 gene, or both. The hRif1 full-length cDNA was cloned into a yeast 2μ-plasmid under the control of the ADH1 promoter and transformed into a RIF1/rif1, RIF2/rif2 heterozygous S. cerevisiae strain. The empty vector was used as a control. The resulting strains were sporulated and spores with desired genotypes overexpressing hRif1 or vector alone were selected and passaged on plates consecutively for five streaks (∼125 population doublings) to allow telomere length to reach equilibrium. We then compared the effects of human Rif1 overexpression in the wild-type, Δrif1, Δrif2 and Δrif1Δrif2 mutant yeast cells. Data from five independent sporulation experiments showed that overexpression of hRif1 in wild-type and Δrif2 strains led to significant telomere elongation; mean telomere length averaged 330 and 910 bp longer, respectively. In contrast, overexpression of hRif1 in Δrif1 or Δrif1Δrif2 strains did not induce significant telomere length changes compared with the vector control (changes ≤120 bp; Fig. 6, A and B; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1). The finding that hRif1 overexpression perturbs telomere length maintenance, in a manner specifically dependent on the presence of the S. cerevisiae Rif1 gene, suggests that hRif1 and scRif1p proteins share at least some conserved functional interactions that can directly or indirectly affect 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