Nucleolar protein PinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA - PubMed (original) (raw)

. 2004 Feb 15;18(4):387-96.

doi: 10.1101/gad.1171804. Epub 2004 Feb 20.

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Nucleolar protein PinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA

Jue Lin et al. Genes Dev. 2004.

Abstract

Human TRF1-binding protein PinX1 inhibits telomerase activity. Here we report that overexpression of yeast PinX1p (yPinX1p) results in shortened telomeres and decreased in vitro telomerase activity. yPinX1p coimmunoprecipitated with yeast telomerase protein Est2p even in cells lacking the telomerase RNA TLC1, or the telomerase-associated proteins Est1p and Est3p. Est2p regions required for binding to yPinX1p or TLC1 were similar. Furthermore, we found two distinct Est2p complexes exist, containing either yPinX1p or TLC1. Levels of Est2p-yPinX1p complex increased when TLC1 was deleted and decreased when TLC1 was overexpressed. Hence, we propose that yPinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.

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Figures

Figure 1.

Figure 1.

Overexpression of yPinX1p leads to shortened telomeres and decreased in vitro telomerase activity. (A) Southern blot of telomeres in wild-type cells (strain BY4705) overexpressing yPinX1p. (B) Δ_rif1_ (yEHB0234) and Δ_rif2_ (yEHB0235) strains overexpressing yPinX1p. (C) Wild-type cells overexpressing both yPinX1p and Est2p. (V) Vector plasmid; (P) yPinX1 overexpression plasmid with Gal promoter (pRS316GalPinX1); (E) Est2myc overexpression plasmid (pRS424Est2myc); (–) cells grown in glucose medium; (+) cells grown in galactose medium. In B, results from two independent transformants with pRS316GalPinX1 are shown. (D) In vitro telomerase activity assay. (LC) Loading control. A γ-32P-ATP-labeled oligonucleotide was added to the assay as the loading control. Products with 1–6 nt added to the primer are indicated.

Figure 2.

Figure 2.

Coimmunoprecipitation of yPinX1p and Est2p. (A) Coimmunoprecipitation of Est2p-myc with yPinX1p-TAP using IgG beads. (B) Coimmunoprecipitation of yPinX1p-myc with Est2p-TAP using IgG beads. For Western blotting (WB), 2% of the extract used for coimmunoprecipitation experiments was loaded in whole-cell extract lanes.

Figure 3.

Figure 3.

Coimmunoprecipitation of yPinX1p and Est2p is independent of TLC1 RNA, Est1p, or Est3p. (A) Overexpressed Est2p-myc copurified with yPinX1p-TAP in strain yEHB4084 containing wild-type TLC1, EST1, and EST3. Est2p-myc was on a 2μ plasmid (pRS424Est2-myc), and yPinX1-TAP was expressed from the Gal promoter on plasmid pRS316GalPinX1-TAP. (B) Est2p-myc copurified with yPinX1p in Δ_tlc1_, Δ_est1_, or Δ_est3_ strains. (S) S100; (B) IgG beads; (E) eluate from IgG beads. Fractions 1–5 are eluate from calmodulin beads. For Western blotting, 0.2% of total extract, 2% of IgG beads after binding, 2% of IgG beads eluate, and 20% of calmodulin eluate were loaded.

Figure 4.

Figure 4.

The same region in Est2p is required for both yPinX1p and TLC1 binding. (A) Diagram of Est2p domain structures. (B) Est2p mutant proteins deleted of domain N, GQ, RT, or C copurify with yPinX1p. Est2p mutant proteins deleted of domain CP, QFP, or T do not copurify with yPinX1p. (S) S100; (B) IgG beads; (E) eluate from IgG beads. Fractions 1–5 are eluate from calmodulin beads. For Western blotting, 0.2% of total extract, 2% of IgG beads after binding, 2% of IgG beads eluate, and 20% of calmodulin eluate were loaded. (C) Est2p mutant proteins deleted of domain CP, QFP, or T do not bind TLC1. (Top panel) Western blotting of Est2p domain deletion mutants. (Lower panel) Northern blotting of TLC1 RNA. (WCE) RNA prepared from whole-cell extracts; (IP) RNA prepared from cell extracts immunoprecipitated with anti-myc antibody 9E10. For RNA preparation, 10% of input whole-cell extract was used and was loaded in WCE lanes.

Figure 5.

Figure 5.

Detection of two distinct Est2p complexes: Est2p–TLC1 and Est2p–yPinX1p. (A) DEAE purified fractions contain TLC1 and Est2p, but no detectable yPinX1p. (S) S100 extract; (FT) flowthrough from DEAE column; (W) last wash from DEAE column; (#1–9) DEAE eluted fractions. (B) yPinX1p-TAP complex purified with IgG contained Est2p, but not TLC1. (S) S100 extract; (FT) flowthrough from IgG beads; (IP) immunoprecipitated samples. (Top panel) Northern blot analysis probed for TLC1 RNA. (Bottom panel) Western blot analysis probed for Est2p-myc. For Western blot analysis and preparation of RNA for Northern blot analysis, 10% of the input extract was used.

Figure 6.

Figure 6.

TLC1 levels influence yPinX1–Est2p binding efficiency. (A) Deletion of TLC1 results in more Est2p associated with yPinX1p, and overexpression of TLC1 results in less Est2p associated with yPinX1p. (O/E) TLC1 overexpressed from the GAL1 promoter. PGK serves as the loading control for Western blotting. The amount of TLC1 RNA is shown on the bottom with actin mRNA as the loading control. (B) Amodel for yPinX1p-mediated inhibition of telomerase activity. See text for description.

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