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Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin.

Gardano L, Holland L, Oulton R, Le Bihan T, Harrington L - Nucleic Acids Res. (2011)

Bottom Line: Telomeres, the ends of linear chromosomes, safeguard against genome instability.One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase.These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3' terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was 'supershifted' with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions.

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Telomerase mobility when purified at low and high stringencies. (A) Analysis of telomerase activity and hTR mobility (hTR, black arrow) by native gel electrophoresis after purification at 0.1 M NaCl (low stringency; LS), and 0.6 M NaCl (high stringency; HS), as described in Figure 1. T (arrow at top) indicates the mobility of thyroglobulin. Lane numbers correspond to gel slices, from top to bottom, as indicated. At right, a dilution of RRL-produced telomerase (1.0 and 0.2 µl, respectively) and no added extract (−) as controls. Asterisk marks the internal PCR standard for the TRAP reaction. The LS profile is the same as shown in Figure 1B. (B) Relative yield of hTR after affinity purification. Bottom panel; RT–PCR analysis using hTR-specific primers with the following amounts of input hTR: 0 (−), 0.1, 1.0, 10, 100, 1000 and 10 000 fg. Top panel: hTR recovery as assessed by RT–PCR of equal volumes of LS + RNase (LSR), LS and HS purifications from the wash (W) and first and second eluates (E1, E2). (C) The same LS and HS eluates as in (B) were resolved via denaturing SDS–PAGE and subjected to Coomassie staining. Protein markers are indicated at left, in kDa. The average protein yield of the affinity eluate (in µg) is indicated below (n = 6, LS ave. 509, SD 73, HS ave. 367, SD 67). (D) Enrichment of hTR relative to hvg1. Top panel: as a control for the ability to detect hvg1, an equivalent amount of hvg1 RNA was added to purified hTR (0, 5, 50, 500, 5000 fg, respectively) or to affinity purified fractions (right, as in B), prior to RT–PCR amplification with hTR-specific and hvg1-specific primers. Bottom panel: affinity purified samples, without the addition of any exogenous RNA, were similarly amplified with hvg1 and hTR primers. Black arrow; hTR, grey arrow; hvg1.
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gkr1243-F2: Telomerase mobility when purified at low and high stringencies. (A) Analysis of telomerase activity and hTR mobility (hTR, black arrow) by native gel electrophoresis after purification at 0.1 M NaCl (low stringency; LS), and 0.6 M NaCl (high stringency; HS), as described in Figure 1. T (arrow at top) indicates the mobility of thyroglobulin. Lane numbers correspond to gel slices, from top to bottom, as indicated. At right, a dilution of RRL-produced telomerase (1.0 and 0.2 µl, respectively) and no added extract (−) as controls. Asterisk marks the internal PCR standard for the TRAP reaction. The LS profile is the same as shown in Figure 1B. (B) Relative yield of hTR after affinity purification. Bottom panel; RT–PCR analysis using hTR-specific primers with the following amounts of input hTR: 0 (−), 0.1, 1.0, 10, 100, 1000 and 10 000 fg. Top panel: hTR recovery as assessed by RT–PCR of equal volumes of LS + RNase (LSR), LS and HS purifications from the wash (W) and first and second eluates (E1, E2). (C) The same LS and HS eluates as in (B) were resolved via denaturing SDS–PAGE and subjected to Coomassie staining. Protein markers are indicated at left, in kDa. The average protein yield of the affinity eluate (in µg) is indicated below (n = 6, LS ave. 509, SD 73, HS ave. 367, SD 67). (D) Enrichment of hTR relative to hvg1. Top panel: as a control for the ability to detect hvg1, an equivalent amount of hvg1 RNA was added to purified hTR (0, 5, 50, 500, 5000 fg, respectively) or to affinity purified fractions (right, as in B), prior to RT–PCR amplification with hTR-specific and hvg1-specific primers. Bottom panel: affinity purified samples, without the addition of any exogenous RNA, were similarly amplified with hvg1 and hTR primers. Black arrow; hTR, grey arrow; hvg1.

Mentions: We next assessed whether various treatments altered the mobility of the endogenous human telomerase complex. Similar to previously published results whereby telomerase mass was estimated by velocity sedimentation or size exclusion chromatography, we found that at low concentrations of NaCl (0.1 M = LS), telomerase activity migrated at an apparent molecular mass >670 kDa (thyroglobulin = T) (Figure 1C). Purification at 37°C (compared with 4°C) did not significantly affect the mobility of telomerase (Figure 1B and C) (63). The faster mobility of hTR after proteinase K treatment (Figure 1B and C), although variable between experiments and not coincident with in vitro purified hTR (compare lower two hTR panels in Figure 1C), nevertheless confirmed that the purified hTR complex contained protein. Upon increasing the stringency of the purification to 0.6 M NaCl the native mobility of human telomerase was increased (Figures 1B and 2A) (17–19,22). Thus, hTR mobility in native gels could be altered by salt concentration, which could reflect distinct properties including protein composition.Figure 2.


Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin.

Gardano L, Holland L, Oulton R, Le Bihan T, Harrington L - Nucleic Acids Res. (2011)

Telomerase mobility when purified at low and high stringencies. (A) Analysis of telomerase activity and hTR mobility (hTR, black arrow) by native gel electrophoresis after purification at 0.1 M NaCl (low stringency; LS), and 0.6 M NaCl (high stringency; HS), as described in Figure 1. T (arrow at top) indicates the mobility of thyroglobulin. Lane numbers correspond to gel slices, from top to bottom, as indicated. At right, a dilution of RRL-produced telomerase (1.0 and 0.2 µl, respectively) and no added extract (−) as controls. Asterisk marks the internal PCR standard for the TRAP reaction. The LS profile is the same as shown in Figure 1B. (B) Relative yield of hTR after affinity purification. Bottom panel; RT–PCR analysis using hTR-specific primers with the following amounts of input hTR: 0 (−), 0.1, 1.0, 10, 100, 1000 and 10 000 fg. Top panel: hTR recovery as assessed by RT–PCR of equal volumes of LS + RNase (LSR), LS and HS purifications from the wash (W) and first and second eluates (E1, E2). (C) The same LS and HS eluates as in (B) were resolved via denaturing SDS–PAGE and subjected to Coomassie staining. Protein markers are indicated at left, in kDa. The average protein yield of the affinity eluate (in µg) is indicated below (n = 6, LS ave. 509, SD 73, HS ave. 367, SD 67). (D) Enrichment of hTR relative to hvg1. Top panel: as a control for the ability to detect hvg1, an equivalent amount of hvg1 RNA was added to purified hTR (0, 5, 50, 500, 5000 fg, respectively) or to affinity purified fractions (right, as in B), prior to RT–PCR amplification with hTR-specific and hvg1-specific primers. Bottom panel: affinity purified samples, without the addition of any exogenous RNA, were similarly amplified with hvg1 and hTR primers. Black arrow; hTR, grey arrow; hvg1.
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Related In: Results  -  Collection

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gkr1243-F2: Telomerase mobility when purified at low and high stringencies. (A) Analysis of telomerase activity and hTR mobility (hTR, black arrow) by native gel electrophoresis after purification at 0.1 M NaCl (low stringency; LS), and 0.6 M NaCl (high stringency; HS), as described in Figure 1. T (arrow at top) indicates the mobility of thyroglobulin. Lane numbers correspond to gel slices, from top to bottom, as indicated. At right, a dilution of RRL-produced telomerase (1.0 and 0.2 µl, respectively) and no added extract (−) as controls. Asterisk marks the internal PCR standard for the TRAP reaction. The LS profile is the same as shown in Figure 1B. (B) Relative yield of hTR after affinity purification. Bottom panel; RT–PCR analysis using hTR-specific primers with the following amounts of input hTR: 0 (−), 0.1, 1.0, 10, 100, 1000 and 10 000 fg. Top panel: hTR recovery as assessed by RT–PCR of equal volumes of LS + RNase (LSR), LS and HS purifications from the wash (W) and first and second eluates (E1, E2). (C) The same LS and HS eluates as in (B) were resolved via denaturing SDS–PAGE and subjected to Coomassie staining. Protein markers are indicated at left, in kDa. The average protein yield of the affinity eluate (in µg) is indicated below (n = 6, LS ave. 509, SD 73, HS ave. 367, SD 67). (D) Enrichment of hTR relative to hvg1. Top panel: as a control for the ability to detect hvg1, an equivalent amount of hvg1 RNA was added to purified hTR (0, 5, 50, 500, 5000 fg, respectively) or to affinity purified fractions (right, as in B), prior to RT–PCR amplification with hTR-specific and hvg1-specific primers. Bottom panel: affinity purified samples, without the addition of any exogenous RNA, were similarly amplified with hvg1 and hTR primers. Black arrow; hTR, grey arrow; hvg1.
Mentions: We next assessed whether various treatments altered the mobility of the endogenous human telomerase complex. Similar to previously published results whereby telomerase mass was estimated by velocity sedimentation or size exclusion chromatography, we found that at low concentrations of NaCl (0.1 M = LS), telomerase activity migrated at an apparent molecular mass >670 kDa (thyroglobulin = T) (Figure 1C). Purification at 37°C (compared with 4°C) did not significantly affect the mobility of telomerase (Figure 1B and C) (63). The faster mobility of hTR after proteinase K treatment (Figure 1B and C), although variable between experiments and not coincident with in vitro purified hTR (compare lower two hTR panels in Figure 1C), nevertheless confirmed that the purified hTR complex contained protein. Upon increasing the stringency of the purification to 0.6 M NaCl the native mobility of human telomerase was increased (Figures 1B and 2A) (17–19,22). Thus, hTR mobility in native gels could be altered by salt concentration, which could reflect distinct properties including protein composition.Figure 2.

Bottom Line: Telomeres, the ends of linear chromosomes, safeguard against genome instability.One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase.These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3' terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was 'supershifted' with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions.

Show MeSH
Related in: MedlinePlus