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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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Sequence conservation and expression of hRif1. (A) Schematic representation of sequence similarities of Rif1 proteins. Percentage identities are in black and percentage similarities are in red. A putative NLS is marked in green. The alternatively spliced-out peptide of hRif1 is marked in yellow. (B) Ubiquitous expression of hRif1 mRNA in human cancer cell lines. 2 μg of mRNAs from different cell lines were treated with glyoxal, electrophoresed on a 20 cm × 25 cm 0.8% agarose gel, transferred to a Hybond NX membrane and hybridized with hRif1 or β-actin cDNA probes. The bracket and arrowhead indicate groups of alternatively spliced hRif1 mRNAs. (C) Expression of hRif1 mRNA in adult human tissues. A multiple tissue Northern blot (CLONTECH Laboratories, Inc.) was hybridized with hRif1 or β-actin cDNA probes. Separation of alternatively spliced hRif1 mRNA was not as clear as in B because the mRNA electrophoresis was performed in a much smaller (6 cm × 8 cm) 1% agarose gel. The bracket and arrowhead indicate the groups of hRif1 mRNAs corresponding to those indicated in B. (D) Immunoblot of hRif1 protein with antibody PAB2857. LOX cells were infected with lentiviruses expressing siRNA sequences targeting hRif1 mRNA. 3 d after infection, 50 μg of whole cell extracts were analyzed by Western blot analysis and probed with PAB2857.
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fig1: Sequence conservation and expression of hRif1. (A) Schematic representation of sequence similarities of Rif1 proteins. Percentage identities are in black and percentage similarities are in red. A putative NLS is marked in green. The alternatively spliced-out peptide of hRif1 is marked in yellow. (B) Ubiquitous expression of hRif1 mRNA in human cancer cell lines. 2 μg of mRNAs from different cell lines were treated with glyoxal, electrophoresed on a 20 cm × 25 cm 0.8% agarose gel, transferred to a Hybond NX membrane and hybridized with hRif1 or β-actin cDNA probes. The bracket and arrowhead indicate groups of alternatively spliced hRif1 mRNAs. (C) Expression of hRif1 mRNA in adult human tissues. A multiple tissue Northern blot (CLONTECH Laboratories, Inc.) was hybridized with hRif1 or β-actin cDNA probes. Separation of alternatively spliced hRif1 mRNA was not as clear as in B because the mRNA electrophoresis was performed in a much smaller (6 cm × 8 cm) 1% agarose gel. The bracket and arrowhead indicate the groups of hRif1 mRNAs corresponding to those indicated in B. (D) Immunoblot of hRif1 protein with antibody PAB2857. LOX cells were infected with lentiviruses expressing siRNA sequences targeting hRif1 mRNA. 3 d after infection, 50 μg of whole cell extracts were analyzed by Western blot analysis and probed with PAB2857.

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)

Sequence conservation and expression of hRif1. (A) Schematic representation of sequence similarities of Rif1 proteins. Percentage identities are in black and percentage similarities are in red. A putative NLS is marked in green. The alternatively spliced-out peptide of hRif1 is marked in yellow. (B) Ubiquitous expression of hRif1 mRNA in human cancer cell lines. 2 μg of mRNAs from different cell lines were treated with glyoxal, electrophoresed on a 20 cm × 25 cm 0.8% agarose gel, transferred to a Hybond NX membrane and hybridized with hRif1 or β-actin cDNA probes. The bracket and arrowhead indicate groups of alternatively spliced hRif1 mRNAs. (C) Expression of hRif1 mRNA in adult human tissues. A multiple tissue Northern blot (CLONTECH Laboratories, Inc.) was hybridized with hRif1 or β-actin cDNA probes. Separation of alternatively spliced hRif1 mRNA was not as clear as in B because the mRNA electrophoresis was performed in a much smaller (6 cm × 8 cm) 1% agarose gel. The bracket and arrowhead indicate the groups of hRif1 mRNAs corresponding to those indicated in B. (D) Immunoblot of hRif1 protein with antibody PAB2857. LOX cells were infected with lentiviruses expressing siRNA sequences targeting hRif1 mRNA. 3 d after infection, 50 μg of whole cell extracts were analyzed by Western blot analysis and probed with PAB2857.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172464&req=5

fig1: Sequence conservation and expression of hRif1. (A) Schematic representation of sequence similarities of Rif1 proteins. Percentage identities are in black and percentage similarities are in red. A putative NLS is marked in green. The alternatively spliced-out peptide of hRif1 is marked in yellow. (B) Ubiquitous expression of hRif1 mRNA in human cancer cell lines. 2 μg of mRNAs from different cell lines were treated with glyoxal, electrophoresed on a 20 cm × 25 cm 0.8% agarose gel, transferred to a Hybond NX membrane and hybridized with hRif1 or β-actin cDNA probes. The bracket and arrowhead indicate groups of alternatively spliced hRif1 mRNAs. (C) Expression of hRif1 mRNA in adult human tissues. A multiple tissue Northern blot (CLONTECH Laboratories, Inc.) was hybridized with hRif1 or β-actin cDNA probes. Separation of alternatively spliced hRif1 mRNA was not as clear as in B because the mRNA electrophoresis was performed in a much smaller (6 cm × 8 cm) 1% agarose gel. The bracket and arrowhead indicate the groups of hRif1 mRNAs corresponding to those indicated in B. (D) Immunoblot of hRif1 protein with antibody PAB2857. LOX cells were infected with lentiviruses expressing siRNA sequences targeting hRif1 mRNA. 3 d after infection, 50 μg of whole cell extracts were analyzed by Western blot analysis and probed with PAB2857.
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