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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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Identification of the hEST1A hTR-binding domain (TRID). (A) Schematic representation of previously assigned hEST1A domains and hEST1A truncations. EST1, EST1 homology domain; TPR, tetratricopeptide repeats; PIN, PilT amino-terminal domain (1). The EST1 and TPR domains of the related SMG7/hEST1C form a 14-3-3 domain with a classic phosphoserine-binding pocket (31). For each polypeptide the association with TRAP activity is indicated [determined in Figure 4; for hEST1A (1–855) association with TRAP activity is not shown (ns)]. TRID stands for hTR Interacting Domain identified in B and C. (B) [32P] 5′-end labeled in vitro transcribed hTR (*hTR) was incubated with in RRL translated [35S] methionine-labeled full-length hEST1A, full-length Flag-hEST1A (FL) and Flag-hEST1A fragments (amino acid fragments are indicated). After immunoprecipitation with α-FLAG antibodies and five washes, samples were separated by 4–20% gradient SDS–PAGE. [35S] and [32P] signals were revealed by analysis on a PhosphoImager. Upon placing a plastic film between the gel and the PhosphoImager screen, only the [32P] signal was detected (lower part). Filled black arrows indicate the presence of the polypeptide in the immunoprecipitated fractions and empty arrows indicate the absence of the polypeptide. (C) Quantification of hTR associated with Flag-hEST1A fragments based on the gel in panel B and at least two additional experiments. The values were corrected by the immunoprecipitation efficiency of the individual polypeptides.
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Figure 3: Identification of the hEST1A hTR-binding domain (TRID). (A) Schematic representation of previously assigned hEST1A domains and hEST1A truncations. EST1, EST1 homology domain; TPR, tetratricopeptide repeats; PIN, PilT amino-terminal domain (1). The EST1 and TPR domains of the related SMG7/hEST1C form a 14-3-3 domain with a classic phosphoserine-binding pocket (31). For each polypeptide the association with TRAP activity is indicated [determined in Figure 4; for hEST1A (1–855) association with TRAP activity is not shown (ns)]. TRID stands for hTR Interacting Domain identified in B and C. (B) [32P] 5′-end labeled in vitro transcribed hTR (*hTR) was incubated with in RRL translated [35S] methionine-labeled full-length hEST1A, full-length Flag-hEST1A (FL) and Flag-hEST1A fragments (amino acid fragments are indicated). After immunoprecipitation with α-FLAG antibodies and five washes, samples were separated by 4–20% gradient SDS–PAGE. [35S] and [32P] signals were revealed by analysis on a PhosphoImager. Upon placing a plastic film between the gel and the PhosphoImager screen, only the [32P] signal was detected (lower part). Filled black arrows indicate the presence of the polypeptide in the immunoprecipitated fractions and empty arrows indicate the absence of the polypeptide. (C) Quantification of hTR associated with Flag-hEST1A fragments based on the gel in panel B and at least two additional experiments. The values were corrected by the immunoprecipitation efficiency of the individual polypeptides.

Mentions: To map the hEST1A domain that mediates hTR binding, FLAG-hEST1A fragments (Figure 3A) were expressed in RRL in presence of [35S]-methionine and incubated with [32P]-labeled hTR. After immunoprecipitation of FLAG-hEST1A with α-FLAG antibodies, samples were separated by SDS–PAGE. [35S]-labeled proteins and [32P]-labeled hTR were detected upon analysis on a PhosphoImager (Figure 3B). [32P]-labeled hTR could be unequivocally distinguished from [35S]-labeled proteins by shielding the lower energy [35S]-derived β-rays with a sheet of plastic (Figure 3B, lower part). In the absence of the FLAG-epitope (lane hEST1A_FL), neither hEST1A nor hTR was detected in the immunoprecipitate, demonstrating the specificity of the assay for FLAG-tagged hEST1A. A FLAG-hEST1A fragment encompassing amino acids 243–412 immunoprecipitated hTR even more efficiently than full-length FLAG-hEST1A, while FLAG-hEST1A fragments lacking this domain did not reveal efficient hTR-binding activity (Figure 3C). We therefore refer to this domain as hTR-interaction domain (TRID).Figure 3.


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

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

Identification of the hEST1A hTR-binding domain (TRID). (A) Schematic representation of previously assigned hEST1A domains and hEST1A truncations. EST1, EST1 homology domain; TPR, tetratricopeptide repeats; PIN, PilT amino-terminal domain (1). The EST1 and TPR domains of the related SMG7/hEST1C form a 14-3-3 domain with a classic phosphoserine-binding pocket (31). For each polypeptide the association with TRAP activity is indicated [determined in Figure 4; for hEST1A (1–855) association with TRAP activity is not shown (ns)]. TRID stands for hTR Interacting Domain identified in B and C. (B) [32P] 5′-end labeled in vitro transcribed hTR (*hTR) was incubated with in RRL translated [35S] methionine-labeled full-length hEST1A, full-length Flag-hEST1A (FL) and Flag-hEST1A fragments (amino acid fragments are indicated). After immunoprecipitation with α-FLAG antibodies and five washes, samples were separated by 4–20% gradient SDS–PAGE. [35S] and [32P] signals were revealed by analysis on a PhosphoImager. Upon placing a plastic film between the gel and the PhosphoImager screen, only the [32P] signal was detected (lower part). Filled black arrows indicate the presence of the polypeptide in the immunoprecipitated fractions and empty arrows indicate the absence of the polypeptide. (C) Quantification of hTR associated with Flag-hEST1A fragments based on the gel in panel B and at least two additional experiments. The values were corrected by the immunoprecipitation efficiency of the individual polypeptides.
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Figure 3: Identification of the hEST1A hTR-binding domain (TRID). (A) Schematic representation of previously assigned hEST1A domains and hEST1A truncations. EST1, EST1 homology domain; TPR, tetratricopeptide repeats; PIN, PilT amino-terminal domain (1). The EST1 and TPR domains of the related SMG7/hEST1C form a 14-3-3 domain with a classic phosphoserine-binding pocket (31). For each polypeptide the association with TRAP activity is indicated [determined in Figure 4; for hEST1A (1–855) association with TRAP activity is not shown (ns)]. TRID stands for hTR Interacting Domain identified in B and C. (B) [32P] 5′-end labeled in vitro transcribed hTR (*hTR) was incubated with in RRL translated [35S] methionine-labeled full-length hEST1A, full-length Flag-hEST1A (FL) and Flag-hEST1A fragments (amino acid fragments are indicated). After immunoprecipitation with α-FLAG antibodies and five washes, samples were separated by 4–20% gradient SDS–PAGE. [35S] and [32P] signals were revealed by analysis on a PhosphoImager. Upon placing a plastic film between the gel and the PhosphoImager screen, only the [32P] signal was detected (lower part). Filled black arrows indicate the presence of the polypeptide in the immunoprecipitated fractions and empty arrows indicate the absence of the polypeptide. (C) Quantification of hTR associated with Flag-hEST1A fragments based on the gel in panel B and at least two additional experiments. The values were corrected by the immunoprecipitation efficiency of the individual polypeptides.
Mentions: To map the hEST1A domain that mediates hTR binding, FLAG-hEST1A fragments (Figure 3A) were expressed in RRL in presence of [35S]-methionine and incubated with [32P]-labeled hTR. After immunoprecipitation of FLAG-hEST1A with α-FLAG antibodies, samples were separated by SDS–PAGE. [35S]-labeled proteins and [32P]-labeled hTR were detected upon analysis on a PhosphoImager (Figure 3B). [32P]-labeled hTR could be unequivocally distinguished from [35S]-labeled proteins by shielding the lower energy [35S]-derived β-rays with a sheet of plastic (Figure 3B, lower part). In the absence of the FLAG-epitope (lane hEST1A_FL), neither hEST1A nor hTR was detected in the immunoprecipitate, demonstrating the specificity of the assay for FLAG-tagged hEST1A. A FLAG-hEST1A fragment encompassing amino acids 243–412 immunoprecipitated hTR even more efficiently than full-length FLAG-hEST1A, while FLAG-hEST1A fragments lacking this domain did not reveal efficient hTR-binding activity (Figure 3C). We therefore refer to this domain as hTR-interaction domain (TRID).Figure 3.

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