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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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Recombinant GST–hEST1A interacts with hTR with high affinity but low specificity. (A) EMSA analysis of [32P] 5′-end labeled in vitro transcribed hTR (*hTR, 1 nM) with increasing amounts of purified GST–hEST1A (fraction E1). Lane 1: *hTR alone. Lane 2: *hTR incubated in EMSA buffer. Lanes 3–12: *hTR incubated with 0.83, 4.15, 8.3, 20.75, 33.2, 41.5, 53.95, 66.4, 83.0 and 166 nM of GST–hEST1A. GST–hEST1A–*hTR complexes are indicated. Right panel: Percentage of *hTR bound to GST–hEST1A plotted as a function of the concentration of GST–hEST1A. The experimental points fitted well with a curve with a Kd of 25 nM. (B) EMSA competition experiment to identify GST–hEST1A-binding specificity for different hTR domains. The secondary structure models are schematically depicted. Lane 1: *hTR alone. Lane 2: B: *hTR incubated with EMSA-buffer. Lanes 3–7: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of unlabeled full-length hTR (5-, 10-, 50-, 100- and 500-fold excess). Lanes 8–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalents of cold DCR45 (5-, 10-, 50-, 100-, 500- and 1000-fold excess). Lanes 14–19: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold Pseudo (5-, 10-, 50-, 100-, 200- and 500-fold excess). Lanes 20–24: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold ΔPseudo (5-, 10-, 50-, 100- and 500-fold excess). Right panel: Quantification of competition experiments. (C) EMSA competition experiment with additional RNA molecules. Lane 1: *hTR incubated with EMSA buffer. Lane 2: *hTR incubated with 50 nM GST–hEST1A (fraction E1). Lanes 3–8: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing mass equivalents of cold full-length hTR (6.25-, 12.5-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Lanes 9–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold gel purified ribosomal RNA (rRNA) (6.25-, 12.5-, 25-, 62.5- and 100-fold mass excess over hTR). Lanes 14–17: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold E. coli tRNA (100-, 250-, 450- and 1800-fold mass excess over hTR). Lanes 18–23: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of heparin (6.25-, 12.25-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Right panel: quantification of competition experiments.
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Figure 2: Recombinant GST–hEST1A interacts with hTR with high affinity but low specificity. (A) EMSA analysis of [32P] 5′-end labeled in vitro transcribed hTR (*hTR, 1 nM) with increasing amounts of purified GST–hEST1A (fraction E1). Lane 1: *hTR alone. Lane 2: *hTR incubated in EMSA buffer. Lanes 3–12: *hTR incubated with 0.83, 4.15, 8.3, 20.75, 33.2, 41.5, 53.95, 66.4, 83.0 and 166 nM of GST–hEST1A. GST–hEST1A–*hTR complexes are indicated. Right panel: Percentage of *hTR bound to GST–hEST1A plotted as a function of the concentration of GST–hEST1A. The experimental points fitted well with a curve with a Kd of 25 nM. (B) EMSA competition experiment to identify GST–hEST1A-binding specificity for different hTR domains. The secondary structure models are schematically depicted. Lane 1: *hTR alone. Lane 2: B: *hTR incubated with EMSA-buffer. Lanes 3–7: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of unlabeled full-length hTR (5-, 10-, 50-, 100- and 500-fold excess). Lanes 8–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalents of cold DCR45 (5-, 10-, 50-, 100-, 500- and 1000-fold excess). Lanes 14–19: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold Pseudo (5-, 10-, 50-, 100-, 200- and 500-fold excess). Lanes 20–24: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold ΔPseudo (5-, 10-, 50-, 100- and 500-fold excess). Right panel: Quantification of competition experiments. (C) EMSA competition experiment with additional RNA molecules. Lane 1: *hTR incubated with EMSA buffer. Lane 2: *hTR incubated with 50 nM GST–hEST1A (fraction E1). Lanes 3–8: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing mass equivalents of cold full-length hTR (6.25-, 12.5-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Lanes 9–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold gel purified ribosomal RNA (rRNA) (6.25-, 12.5-, 25-, 62.5- and 100-fold mass excess over hTR). Lanes 14–17: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold E. coli tRNA (100-, 250-, 450- and 1800-fold mass excess over hTR). Lanes 18–23: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of heparin (6.25-, 12.25-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Right panel: quantification of competition experiments.

Mentions: Association of hEST1A with telomerase may be mediated by protein–protein and/or protein–RNA interactions. In order to test the ability of hEST1A to bind the telomerase RNA moiety hTR, RNA band shift experiments were carried out (Figure 2). Increasing amounts of GST–hEST1A were incubated with [32P]-5′-end labeled in vitro transcribed hTR. Separation of the fractions on non-denaturing polyacrylamide gels indicated formation of slower migrating GST–hEST1A–hTR complexes (Figure 2A). The experimental data for overall hEST1A–hTR-binding fitted best a theoretical curve with a Kd of 25 nM (Figure 2A). The spread-out appearance of the GST–hEST1A–hTR complexes may be due to conformational flexibility, complex heterogeneity and concomitant binding of several hEST1A polypeptides per hTR molecule (see below). Thus this analysis gives an estimate for the overall Kd of hEST1A–hTR complexes without distinguishing individual subtypes. Other gel recipes, removal of the GST-tag from hEST1A or the use of hTR fragments did not reduce the heterogeneity. To test binding affinity for different domains of hTR, different fragments of hTR were transcribed in vitro and tested for their ability to inhibit the formation of the full-length GST–hEST1A–hTR complex (Figure 2B). The Δ-Pseudo fragment had a similar capacity as unlabeled full-length hTR, to compete for the formation of the [32P]-labeled full-length GST–hEST1A–hTR complex (Figure 2B). However, two other hTR fragments (Pseudo and ΔCR45) also competed well with hTR for hEST1A-binding at slightly higher concentrations. Since the three tested hTR fragments contained distinct non-overlapping domains, this indicated several hEST1A–hTR interaction sites. To further address specificity of RNA binding, other non-related RNAs were tested in a competition experiment (Figure 2C). This experiment indicated that the affinity of hEST1A for rRNA is similar as for hTR, while tRNA competes only at ∼20-fold higher concentration, indicating very low affinity of hEST1A for this RNA. Heparin efficiently inhibited hTR binding.Figure 2.


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

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

Recombinant GST–hEST1A interacts with hTR with high affinity but low specificity. (A) EMSA analysis of [32P] 5′-end labeled in vitro transcribed hTR (*hTR, 1 nM) with increasing amounts of purified GST–hEST1A (fraction E1). Lane 1: *hTR alone. Lane 2: *hTR incubated in EMSA buffer. Lanes 3–12: *hTR incubated with 0.83, 4.15, 8.3, 20.75, 33.2, 41.5, 53.95, 66.4, 83.0 and 166 nM of GST–hEST1A. GST–hEST1A–*hTR complexes are indicated. Right panel: Percentage of *hTR bound to GST–hEST1A plotted as a function of the concentration of GST–hEST1A. The experimental points fitted well with a curve with a Kd of 25 nM. (B) EMSA competition experiment to identify GST–hEST1A-binding specificity for different hTR domains. The secondary structure models are schematically depicted. Lane 1: *hTR alone. Lane 2: B: *hTR incubated with EMSA-buffer. Lanes 3–7: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of unlabeled full-length hTR (5-, 10-, 50-, 100- and 500-fold excess). Lanes 8–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalents of cold DCR45 (5-, 10-, 50-, 100-, 500- and 1000-fold excess). Lanes 14–19: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold Pseudo (5-, 10-, 50-, 100-, 200- and 500-fold excess). Lanes 20–24: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold ΔPseudo (5-, 10-, 50-, 100- and 500-fold excess). Right panel: Quantification of competition experiments. (C) EMSA competition experiment with additional RNA molecules. Lane 1: *hTR incubated with EMSA buffer. Lane 2: *hTR incubated with 50 nM GST–hEST1A (fraction E1). Lanes 3–8: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing mass equivalents of cold full-length hTR (6.25-, 12.5-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Lanes 9–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold gel purified ribosomal RNA (rRNA) (6.25-, 12.5-, 25-, 62.5- and 100-fold mass excess over hTR). Lanes 14–17: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold E. coli tRNA (100-, 250-, 450- and 1800-fold mass excess over hTR). Lanes 18–23: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of heparin (6.25-, 12.25-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Right panel: quantification of competition experiments.
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Figure 2: Recombinant GST–hEST1A interacts with hTR with high affinity but low specificity. (A) EMSA analysis of [32P] 5′-end labeled in vitro transcribed hTR (*hTR, 1 nM) with increasing amounts of purified GST–hEST1A (fraction E1). Lane 1: *hTR alone. Lane 2: *hTR incubated in EMSA buffer. Lanes 3–12: *hTR incubated with 0.83, 4.15, 8.3, 20.75, 33.2, 41.5, 53.95, 66.4, 83.0 and 166 nM of GST–hEST1A. GST–hEST1A–*hTR complexes are indicated. Right panel: Percentage of *hTR bound to GST–hEST1A plotted as a function of the concentration of GST–hEST1A. The experimental points fitted well with a curve with a Kd of 25 nM. (B) EMSA competition experiment to identify GST–hEST1A-binding specificity for different hTR domains. The secondary structure models are schematically depicted. Lane 1: *hTR alone. Lane 2: B: *hTR incubated with EMSA-buffer. Lanes 3–7: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of unlabeled full-length hTR (5-, 10-, 50-, 100- and 500-fold excess). Lanes 8–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalents of cold DCR45 (5-, 10-, 50-, 100-, 500- and 1000-fold excess). Lanes 14–19: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold Pseudo (5-, 10-, 50-, 100-, 200- and 500-fold excess). Lanes 20–24: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing molar equivalent of cold ΔPseudo (5-, 10-, 50-, 100- and 500-fold excess). Right panel: Quantification of competition experiments. (C) EMSA competition experiment with additional RNA molecules. Lane 1: *hTR incubated with EMSA buffer. Lane 2: *hTR incubated with 50 nM GST–hEST1A (fraction E1). Lanes 3–8: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing mass equivalents of cold full-length hTR (6.25-, 12.5-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Lanes 9–13: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold gel purified ribosomal RNA (rRNA) (6.25-, 12.5-, 25-, 62.5- and 100-fold mass excess over hTR). Lanes 14–17: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of cold E. coli tRNA (100-, 250-, 450- and 1800-fold mass excess over hTR). Lanes 18–23: 50 nM GST–hEST1A (fraction E1) incubated with *hTR and increasing quantity of heparin (6.25-, 12.25-, 25-, 62.5-, 100- and 250-fold mass excess over hTR). Right panel: quantification of competition experiments.
Mentions: Association of hEST1A with telomerase may be mediated by protein–protein and/or protein–RNA interactions. In order to test the ability of hEST1A to bind the telomerase RNA moiety hTR, RNA band shift experiments were carried out (Figure 2). Increasing amounts of GST–hEST1A were incubated with [32P]-5′-end labeled in vitro transcribed hTR. Separation of the fractions on non-denaturing polyacrylamide gels indicated formation of slower migrating GST–hEST1A–hTR complexes (Figure 2A). The experimental data for overall hEST1A–hTR-binding fitted best a theoretical curve with a Kd of 25 nM (Figure 2A). The spread-out appearance of the GST–hEST1A–hTR complexes may be due to conformational flexibility, complex heterogeneity and concomitant binding of several hEST1A polypeptides per hTR molecule (see below). Thus this analysis gives an estimate for the overall Kd of hEST1A–hTR complexes without distinguishing individual subtypes. Other gel recipes, removal of the GST-tag from hEST1A or the use of hTR fragments did not reduce the heterogeneity. To test binding affinity for different domains of hTR, different fragments of hTR were transcribed in vitro and tested for their ability to inhibit the formation of the full-length GST–hEST1A–hTR complex (Figure 2B). The Δ-Pseudo fragment had a similar capacity as unlabeled full-length hTR, to compete for the formation of the [32P]-labeled full-length GST–hEST1A–hTR complex (Figure 2B). However, two other hTR fragments (Pseudo and ΔCR45) also competed well with hTR for hEST1A-binding at slightly higher concentrations. Since the three tested hTR fragments contained distinct non-overlapping domains, this indicated several hEST1A–hTR interaction sites. To further address specificity of RNA binding, other non-related RNAs were tested in a competition experiment (Figure 2C). This experiment indicated that the affinity of hEST1A for rRNA is similar as for hTR, while tRNA competes only at ∼20-fold higher concentration, indicating very low affinity of hEST1A for this RNA. Heparin efficiently inhibited hTR binding.Figure 2.

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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