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Protein RNA and protein protein interactions mediate association of human EST1A/SMG6 with telomerase.

Redon S, Reichenbach P, Lingner J - Nucleic Acids Res. (2007)

Bottom Line: Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity.Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K(d) = 25 nM) but low specificity.We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein-protein contacts with hTERT.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne (EPFL) and National Center of Competence in Research Frontiers in Genetics, CH-1066 Epalinges s/Lausanne, Switzerland.

ABSTRACT
The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein-RNA and protein-protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein-protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K(d) = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein-protein contacts with hTERT.

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Expression and purification of recombinant GST–hEST1A protein and interaction with in vitro reconstituted telomerase. (A) SDS–PAGE and Coomassie blue staining of GST–hEST1A fractions derived from Hi5 insect cells. Lane 1: MW: Molecular weight markers. Lane 2: L: GST–hEST1A expressing Hi5 nuclear extract. Lane 3: FT: flow through of GSH-beads. Lanes 4–6: E1, E2 and E3: first, second and third eluates, Lanes 7–10: BSA dilutions. GST–hEST1A is indicated. (B) Western blots with anti-GST and anti-hEST1A antibodies. (C) Relative TRAP activity (%) pulled down with GSH-beads upon incubation of 50 nM GST (white rectangles) or 50 nM GST–hEST1A (fraction E1; black rectangles) with telomerase reconstituted in vitro in insect cell lysates (Baculovirus) or in RRL. Interaction of GST–hEST1A and GST with telomerase in vitro. Bars represent the means ± SD of three independent experiments.
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Figure 1: Expression and purification of recombinant GST–hEST1A protein and interaction with in vitro reconstituted telomerase. (A) SDS–PAGE and Coomassie blue staining of GST–hEST1A fractions derived from Hi5 insect cells. Lane 1: MW: Molecular weight markers. Lane 2: L: GST–hEST1A expressing Hi5 nuclear extract. Lane 3: FT: flow through of GSH-beads. Lanes 4–6: E1, E2 and E3: first, second and third eluates, Lanes 7–10: BSA dilutions. GST–hEST1A is indicated. (B) Western blots with anti-GST and anti-hEST1A antibodies. (C) Relative TRAP activity (%) pulled down with GSH-beads upon incubation of 50 nM GST (white rectangles) or 50 nM GST–hEST1A (fraction E1; black rectangles) with telomerase reconstituted in vitro in insect cell lysates (Baculovirus) or in RRL. Interaction of GST–hEST1A and GST with telomerase in vitro. Bars represent the means ± SD of three independent experiments.

Mentions: 1 µl of reconstituted and affinity purified telomerase from insect cells (17) or in rabbit reticulocyte lysates (RRL) (Promega) reconstituted telomerase (19) was incubated with 0.5 pmol of purified GST–hEST1A or GST for 1 h at 4°C in 10 µl final volume of 20 mM HEPES pH 7.9, 100 mM KCl, 1 mM DTT, 10% glycerol and 0.2% Tween 20. 9 µl of this mixture was added to 36 µl of 30% slurry of GSH-Sepharose beads (GE Healthcare) in IP buffer (20 mM HEPES pH 7.9, 100 mM KCl, 1 mM DTT, 10% glycerol, 0.2% Tween 20) and incubated 1 h at 4°C on a wheel. The beads were washed three times with 150 µl of IP buffer. In Figure 1, telomerase activity was measured by TRAP using quantitative PCR and the light cycler (Roche) (18).Figure 1.


Protein RNA and protein protein interactions mediate association of human EST1A/SMG6 with telomerase.

Redon S, Reichenbach P, Lingner J - Nucleic Acids Res. (2007)

Expression and purification of recombinant GST–hEST1A protein and interaction with in vitro reconstituted telomerase. (A) SDS–PAGE and Coomassie blue staining of GST–hEST1A fractions derived from Hi5 insect cells. Lane 1: MW: Molecular weight markers. Lane 2: L: GST–hEST1A expressing Hi5 nuclear extract. Lane 3: FT: flow through of GSH-beads. Lanes 4–6: E1, E2 and E3: first, second and third eluates, Lanes 7–10: BSA dilutions. GST–hEST1A is indicated. (B) Western blots with anti-GST and anti-hEST1A antibodies. (C) Relative TRAP activity (%) pulled down with GSH-beads upon incubation of 50 nM GST (white rectangles) or 50 nM GST–hEST1A (fraction E1; black rectangles) with telomerase reconstituted in vitro in insect cell lysates (Baculovirus) or in RRL. Interaction of GST–hEST1A and GST with telomerase in vitro. Bars represent the means ± SD of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175296&req=5

Figure 1: Expression and purification of recombinant GST–hEST1A protein and interaction with in vitro reconstituted telomerase. (A) SDS–PAGE and Coomassie blue staining of GST–hEST1A fractions derived from Hi5 insect cells. Lane 1: MW: Molecular weight markers. Lane 2: L: GST–hEST1A expressing Hi5 nuclear extract. Lane 3: FT: flow through of GSH-beads. Lanes 4–6: E1, E2 and E3: first, second and third eluates, Lanes 7–10: BSA dilutions. GST–hEST1A is indicated. (B) Western blots with anti-GST and anti-hEST1A antibodies. (C) Relative TRAP activity (%) pulled down with GSH-beads upon incubation of 50 nM GST (white rectangles) or 50 nM GST–hEST1A (fraction E1; black rectangles) with telomerase reconstituted in vitro in insect cell lysates (Baculovirus) or in RRL. Interaction of GST–hEST1A and GST with telomerase in vitro. Bars represent the means ± SD of three independent experiments.
Mentions: 1 µl of reconstituted and affinity purified telomerase from insect cells (17) or in rabbit reticulocyte lysates (RRL) (Promega) reconstituted telomerase (19) was incubated with 0.5 pmol of purified GST–hEST1A or GST for 1 h at 4°C in 10 µl final volume of 20 mM HEPES pH 7.9, 100 mM KCl, 1 mM DTT, 10% glycerol and 0.2% Tween 20. 9 µl of this mixture was added to 36 µl of 30% slurry of GSH-Sepharose beads (GE Healthcare) in IP buffer (20 mM HEPES pH 7.9, 100 mM KCl, 1 mM DTT, 10% glycerol, 0.2% Tween 20) and incubated 1 h at 4°C on a wheel. The beads were washed three times with 150 µl of IP buffer. In Figure 1, telomerase activity was measured by TRAP using quantitative PCR and the light cycler (Roche) (18).Figure 1.

Bottom Line: Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity.Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K(d) = 25 nM) but low specificity.We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein-protein contacts with hTERT.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne (EPFL) and National Center of Competence in Research Frontiers in Genetics, CH-1066 Epalinges s/Lausanne, Switzerland.

ABSTRACT
The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein-RNA and protein-protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein-protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K(d) = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein-protein contacts with hTERT.

Show MeSH
Related in: MedlinePlus