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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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Protein–RNA and protein–protein interactions mediate association of human EST1A with hTERT. The indicated polypeptides were transiently co-expressed in 293T (Figure 7A and B) or WI38-VA13 (Figure 7D) cells and immunoprecipitated (IP) with the indicated antibodies. Polypeptides were revealed by western blotting with antibodies indicated on the left. (A) 293T cell extracts (600 µl) were either treated (+) or not treated (−) with 3 µg RNase A for 10 min at 25°C before starting the immunoprecipitation with α-FLAG antibody. After five washes, samples were loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Myc-EID was determined by western blotting with α-Myc antibodies and the immunoprecipitation efficiency was determined by western blotting with α-FLAG antibodies. (B) Transient co-transfection in 293T cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies. After five washes, the beads were either treated (+) or not treated (−) or with 0.5 µg RNase A/12 µl for 10 min at 25°C. The eluate (E) was also analyzed to check for the release of Flag-hEST1A. The beads were washed one time before being loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Flag-hEST1A was determined by western blotting with α-FLAG antibodies and the immunoprecipitation efficiency was determined by western blotting with α-Myc antibodies. (C) Dot blot analysis of total RNA extracted from 293T cell extracts before and after RNase A treatment, revealed with a randomly labeled full-length hTR probe. Extracts (Input) were treated with 0.005 µg RNase A/µl of extract and beads (IP) were treated with 0.04 µg of RNase A/µl of bead suspension as in B. (D) Transient co-transfection in WI38-VA13 cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies (left panel) or α-Flag antibodies (right panel) as in A and B.
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Figure 7: Protein–RNA and protein–protein interactions mediate association of human EST1A with hTERT. The indicated polypeptides were transiently co-expressed in 293T (Figure 7A and B) or WI38-VA13 (Figure 7D) cells and immunoprecipitated (IP) with the indicated antibodies. Polypeptides were revealed by western blotting with antibodies indicated on the left. (A) 293T cell extracts (600 µl) were either treated (+) or not treated (−) with 3 µg RNase A for 10 min at 25°C before starting the immunoprecipitation with α-FLAG antibody. After five washes, samples were loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Myc-EID was determined by western blotting with α-Myc antibodies and the immunoprecipitation efficiency was determined by western blotting with α-FLAG antibodies. (B) Transient co-transfection in 293T cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies. After five washes, the beads were either treated (+) or not treated (−) or with 0.5 µg RNase A/12 µl for 10 min at 25°C. The eluate (E) was also analyzed to check for the release of Flag-hEST1A. The beads were washed one time before being loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Flag-hEST1A was determined by western blotting with α-FLAG antibodies and the immunoprecipitation efficiency was determined by western blotting with α-Myc antibodies. (C) Dot blot analysis of total RNA extracted from 293T cell extracts before and after RNase A treatment, revealed with a randomly labeled full-length hTR probe. Extracts (Input) were treated with 0.005 µg RNase A/µl of extract and beads (IP) were treated with 0.04 µg of RNase A/µl of bead suspension as in B. (D) Transient co-transfection in WI38-VA13 cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies (left panel) or α-Flag antibodies (right panel) as in A and B.

Mentions: The above experiments delineated hEST1A and hTERT domains that at the same time mediated the interaction between hEST1A and hTERT as well as with hTR. To test the hypothesis that the interaction between hEST1A and hTERT was mediated by hTR, we transiently co-expressed hEST1A and hTERT fragments in 293T cells and tested their interaction by co-immunoprecipitation upon treatment of the extracts with RNase A (Figure 7). The extent of hTR destruction by RNase A treatment was determined by assessing presence of hTR upon Northern hybridization of dotblots with a randomly labeled full-length hTR probe (Figure 7C). Approximately 99% of the hTR signal disappeared in extracts and more than 95% of the signal disappeared in the immunoprecipitates upon RNase A treatment. Immunoprecipitated full-length FLAG-hEST1A co-immunoprecipitated Myc-EID (hTERT-fragment 147–311) and this interaction was not alleviated upon RNase A treatment (Figure 7A, compare lanes 5 and 6 on the right panel). Thus, while EID is able to bind hTR it establishes also protein–protein contacts with hEST1A. In contrast, when a TRID-containing FLAG-hEST1A fragment [hEST1A-fragment (243–533)] was immunoprecipitated, the interaction with EID was RNase A sensitive (Figure 7A, compare lanes 7 and 8 on the right panel). Thus, EID cannot establish stable protein–protein contacts with TRID as observed with full-length hEST1A. Consistent with these results, when either full-length Myc-hTERT or Myc-EID were co-expressed with full-length FLAG-hEST1A and immunoprecipitated, FLAG-hEST1A remained associated with Myc-hTERT as well as with Myc-EID even upon RNase A treatment of the beads after the immunoprecipitation (Figure 7B, compare in the right panel lanes 5 with 7 and lanes 9 with 11).Figure 7.


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

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

Protein–RNA and protein–protein interactions mediate association of human EST1A with hTERT. The indicated polypeptides were transiently co-expressed in 293T (Figure 7A and B) or WI38-VA13 (Figure 7D) cells and immunoprecipitated (IP) with the indicated antibodies. Polypeptides were revealed by western blotting with antibodies indicated on the left. (A) 293T cell extracts (600 µl) were either treated (+) or not treated (−) with 3 µg RNase A for 10 min at 25°C before starting the immunoprecipitation with α-FLAG antibody. After five washes, samples were loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Myc-EID was determined by western blotting with α-Myc antibodies and the immunoprecipitation efficiency was determined by western blotting with α-FLAG antibodies. (B) Transient co-transfection in 293T cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies. After five washes, the beads were either treated (+) or not treated (−) or with 0.5 µg RNase A/12 µl for 10 min at 25°C. The eluate (E) was also analyzed to check for the release of Flag-hEST1A. The beads were washed one time before being loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Flag-hEST1A was determined by western blotting with α-FLAG antibodies and the immunoprecipitation efficiency was determined by western blotting with α-Myc antibodies. (C) Dot blot analysis of total RNA extracted from 293T cell extracts before and after RNase A treatment, revealed with a randomly labeled full-length hTR probe. Extracts (Input) were treated with 0.005 µg RNase A/µl of extract and beads (IP) were treated with 0.04 µg of RNase A/µl of bead suspension as in B. (D) Transient co-transfection in WI38-VA13 cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies (left panel) or α-Flag antibodies (right panel) as in A and B.
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Figure 7: Protein–RNA and protein–protein interactions mediate association of human EST1A with hTERT. The indicated polypeptides were transiently co-expressed in 293T (Figure 7A and B) or WI38-VA13 (Figure 7D) cells and immunoprecipitated (IP) with the indicated antibodies. Polypeptides were revealed by western blotting with antibodies indicated on the left. (A) 293T cell extracts (600 µl) were either treated (+) or not treated (−) with 3 µg RNase A for 10 min at 25°C before starting the immunoprecipitation with α-FLAG antibody. After five washes, samples were loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Myc-EID was determined by western blotting with α-Myc antibodies and the immunoprecipitation efficiency was determined by western blotting with α-FLAG antibodies. (B) Transient co-transfection in 293T cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies. After five washes, the beads were either treated (+) or not treated (−) or with 0.5 µg RNase A/12 µl for 10 min at 25°C. The eluate (E) was also analyzed to check for the release of Flag-hEST1A. The beads were washed one time before being loaded on 4–20% gradient SDS–polyacrylamide gels. The presence of Flag-hEST1A was determined by western blotting with α-FLAG antibodies and the immunoprecipitation efficiency was determined by western blotting with α-Myc antibodies. (C) Dot blot analysis of total RNA extracted from 293T cell extracts before and after RNase A treatment, revealed with a randomly labeled full-length hTR probe. Extracts (Input) were treated with 0.005 µg RNase A/µl of extract and beads (IP) were treated with 0.04 µg of RNase A/µl of bead suspension as in B. (D) Transient co-transfection in WI38-VA13 cells of Flag-hEST1A and full-length FLAG-hTERT, full-length Myc-hTERT (FL) and Myc-EID. Immunoprecipitations were carried out with α-Myc antibodies (left panel) or α-Flag antibodies (right panel) as in A and B.
Mentions: The above experiments delineated hEST1A and hTERT domains that at the same time mediated the interaction between hEST1A and hTERT as well as with hTR. To test the hypothesis that the interaction between hEST1A and hTERT was mediated by hTR, we transiently co-expressed hEST1A and hTERT fragments in 293T cells and tested their interaction by co-immunoprecipitation upon treatment of the extracts with RNase A (Figure 7). The extent of hTR destruction by RNase A treatment was determined by assessing presence of hTR upon Northern hybridization of dotblots with a randomly labeled full-length hTR probe (Figure 7C). Approximately 99% of the hTR signal disappeared in extracts and more than 95% of the signal disappeared in the immunoprecipitates upon RNase A treatment. Immunoprecipitated full-length FLAG-hEST1A co-immunoprecipitated Myc-EID (hTERT-fragment 147–311) and this interaction was not alleviated upon RNase A treatment (Figure 7A, compare lanes 5 and 6 on the right panel). Thus, while EID is able to bind hTR it establishes also protein–protein contacts with hEST1A. In contrast, when a TRID-containing FLAG-hEST1A fragment [hEST1A-fragment (243–533)] was immunoprecipitated, the interaction with EID was RNase A sensitive (Figure 7A, compare lanes 7 and 8 on the right panel). Thus, EID cannot establish stable protein–protein contacts with TRID as observed with full-length hEST1A. Consistent with these results, when either full-length Myc-hTERT or Myc-EID were co-expressed with full-length FLAG-hEST1A and immunoprecipitated, FLAG-hEST1A remained associated with Myc-hTERT as well as with Myc-EID even upon RNase A treatment of the beads after the immunoprecipitation (Figure 7B, compare in the right panel lanes 5 with 7 and lanes 9 with 11).Figure 7.

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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