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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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Impaired cell growth upon depletion of hRif1. (A) Knockdown of hRif1 mRNA levels by siRNA treatment. LOX cells were infected with lentiviruses expressing hairpin siRNAs against different sequences within the protein coding region of hRif1 mRNA. 2 d after infection, 40 μg of total RNA from each cell line was hybridized with hRif1 or GAPDH probes. Relative hRif1 levels were determined by ImageQuant software using GAPDH as the loading control. (B) Depletion of hRif1 protein levels by siRNA. 50 μg of whole cell extracts from each cell line were probed with PAB2857 and α-tubulin antibodies. siRNA treatment was performed as described in A. (C) hRif1 depletion leads to cell growth inhibition in HT1080 and LOX cells. HT1080 cells and LOX cells were infected with lentiviral siRNA constructs at >95% efficiencies, as indicated by a GFP expressed from the same lentiviral vector. Cell numbers were counted every 3 d after infection and cell counts are shown as mean ± SD of plates analyzed in triplicate.
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fig2: Impaired cell growth upon depletion of hRif1. (A) Knockdown of hRif1 mRNA levels by siRNA treatment. LOX cells were infected with lentiviruses expressing hairpin siRNAs against different sequences within the protein coding region of hRif1 mRNA. 2 d after infection, 40 μg of total RNA from each cell line was hybridized with hRif1 or GAPDH probes. Relative hRif1 levels were determined by ImageQuant software using GAPDH as the loading control. (B) Depletion of hRif1 protein levels by siRNA. 50 μg of whole cell extracts from each cell line were probed with PAB2857 and α-tubulin antibodies. siRNA treatment was performed as described in A. (C) hRif1 depletion leads to cell growth inhibition in HT1080 and LOX cells. HT1080 cells and LOX cells were infected with lentiviral siRNA constructs at >95% efficiencies, as indicated by a GFP expressed from the same lentiviral vector. Cell numbers were counted every 3 d after infection and cell counts are shown as mean ± SD of plates analyzed in triplicate.

Mentions: We identified a human orthologue of the yeast telomeric protein Rif1 (Fig. 1 A). A partial human EST sequence that shares sequence homology with the NH2-terminal region of the S. cerevisiae and S. pombe Rif1p was reported previously (Kanoh and Ishikawa, 2001). We assembled a full-length hRif1 cDNA by homology alignment with the partial sequence, exon prediction from genomic sequences, and 5′- and 3′-rapid amplification of cDNA ends (RACE). The full-length cDNA was then cloned by RT-PCR. Multiple alternatively spliced forms of the 5′-untranslated region were detected by the 5′-RACE analyses. However, they all conform to the same initiator ATG sequence. The ORF of the cDNAs encodes a protein of 2,472 aa. A COOH-terminal region of the cDNA encoding 26 aa is alternatively spliced out, resulting in a 2446–amino acid form (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1). RT-PCR from different human cancer cell lines demonstrated that the mRNA encoding this shorter protein product was much more abundant than that encoding the longer product (unpublished data). Sequence analysis of the hRif1 protein shows that it shares sequence homology to S. cerevisiae and S. pombe Rif1 at both NH2- and COOH-terminal regions (Fig. 1 A) but contains no known protein motifs. Northern blotting analysis showed that the hRif1 mRNA is ubiquitously expressed and alternatively spliced in various human cancer cell lines, but the major size differences of alternatively spliced mRNA all occurred outside the protein coding region (Fig. 1 B and Fig. 2 A). Multiple-tissue Northern blots demonstrated that the hRif1 mRNA is highly expressed in testis but is at very low levels in thymus and uterus (Fig. 1 C), which is consistent with the expression pattern of a mouse Rif1 orthologue reported recently (Adams and McLaren, 2004).


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

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

Impaired cell growth upon depletion of hRif1. (A) Knockdown of hRif1 mRNA levels by siRNA treatment. LOX cells were infected with lentiviruses expressing hairpin siRNAs against different sequences within the protein coding region of hRif1 mRNA. 2 d after infection, 40 μg of total RNA from each cell line was hybridized with hRif1 or GAPDH probes. Relative hRif1 levels were determined by ImageQuant software using GAPDH as the loading control. (B) Depletion of hRif1 protein levels by siRNA. 50 μg of whole cell extracts from each cell line were probed with PAB2857 and α-tubulin antibodies. siRNA treatment was performed as described in A. (C) hRif1 depletion leads to cell growth inhibition in HT1080 and LOX cells. HT1080 cells and LOX cells were infected with lentiviral siRNA constructs at >95% efficiencies, as indicated by a GFP expressed from the same lentiviral vector. Cell numbers were counted every 3 d after infection and cell counts are shown as mean ± SD of plates analyzed in triplicate.
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Related In: Results  -  Collection

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fig2: Impaired cell growth upon depletion of hRif1. (A) Knockdown of hRif1 mRNA levels by siRNA treatment. LOX cells were infected with lentiviruses expressing hairpin siRNAs against different sequences within the protein coding region of hRif1 mRNA. 2 d after infection, 40 μg of total RNA from each cell line was hybridized with hRif1 or GAPDH probes. Relative hRif1 levels were determined by ImageQuant software using GAPDH as the loading control. (B) Depletion of hRif1 protein levels by siRNA. 50 μg of whole cell extracts from each cell line were probed with PAB2857 and α-tubulin antibodies. siRNA treatment was performed as described in A. (C) hRif1 depletion leads to cell growth inhibition in HT1080 and LOX cells. HT1080 cells and LOX cells were infected with lentiviral siRNA constructs at >95% efficiencies, as indicated by a GFP expressed from the same lentiviral vector. Cell numbers were counted every 3 d after infection and cell counts are shown as mean ± SD of plates analyzed in triplicate.
Mentions: We identified a human orthologue of the yeast telomeric protein Rif1 (Fig. 1 A). A partial human EST sequence that shares sequence homology with the NH2-terminal region of the S. cerevisiae and S. pombe Rif1p was reported previously (Kanoh and Ishikawa, 2001). We assembled a full-length hRif1 cDNA by homology alignment with the partial sequence, exon prediction from genomic sequences, and 5′- and 3′-rapid amplification of cDNA ends (RACE). The full-length cDNA was then cloned by RT-PCR. Multiple alternatively spliced forms of the 5′-untranslated region were detected by the 5′-RACE analyses. However, they all conform to the same initiator ATG sequence. The ORF of the cDNAs encodes a protein of 2,472 aa. A COOH-terminal region of the cDNA encoding 26 aa is alternatively spliced out, resulting in a 2446–amino acid form (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1). RT-PCR from different human cancer cell lines demonstrated that the mRNA encoding this shorter protein product was much more abundant than that encoding the longer product (unpublished data). Sequence analysis of the hRif1 protein shows that it shares sequence homology to S. cerevisiae and S. pombe Rif1 at both NH2- and COOH-terminal regions (Fig. 1 A) but contains no known protein motifs. Northern blotting analysis showed that the hRif1 mRNA is ubiquitously expressed and alternatively spliced in various human cancer cell lines, but the major size differences of alternatively spliced mRNA all occurred outside the protein coding region (Fig. 1 B and Fig. 2 A). Multiple-tissue Northern blots demonstrated that the hRif1 mRNA is highly expressed in testis but is at very low levels in thymus and uterus (Fig. 1 C), which is consistent with the expression pattern of a mouse Rif1 orthologue reported recently (Adams and McLaren, 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.

Show MeSH
Related in: MedlinePlus