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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 purification via affinity selection and native gel electrophoresis. (A) Co-migration of hTR and telomerase activity after native gel electrophoresis of affinity-purified Raji cell extract (at 0.1 M NaCl). After clear native gel electrophoresis, one lane was stained with Coomassie blue (top panel; gel slice oriented from top to bottom as indicated), and one lane probed with a radiolabelled probe corresponding to hTR after transfer to nylon (middle panel). A third lane was sliced into 13 fractions, eluted into buffer and subjected to the telomere repeat amplification protocol, TRAP (bottom panel). (B) Relative mobility of telomerase after native gel electrophoresis and detection with a radiolabelled probe corresponding to hTR. Lane 1, in vitro transcribed hTR alone; Raji cell extracts purified at 0.1 M NaCl (low stringency; LS) and 0.6 M NaCl (high stringency; HS) at 4°C (lanes 2 and 3, respectively), 37°C (lanes 4 and 6) and after incubation with proteinase K (lanes 5 and 7). Brackets at right indicate position of gel slices. (C) The indicated treatments as in (B) were resolved by native gel electrophoresis, sliced into 13 fractions and subjected to TRAP (upper panels) and RT–PCR analysis to detect the native mobility of hTR (lower panels). Thyroglobulin (T) was loaded onto an adjacent lane of the native gel; its position is indicated with an arrow, top. Asterisk indicates TRAP internal PCR standard; arrows at right, hTR. The bottom panel represents PCR detection of hTR after native gel electrophoresis of purified, in vitro transcribed hTR alone (no added extract). At right, 1.0 and 0.2 µl of rabbit reticulocyte lysate (RRL) containing reconstituted telomerase activity, or water alone (−) as positive and negative controls for TRAP, respectively.
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gkr1243-F1: Telomerase purification via affinity selection and native gel electrophoresis. (A) Co-migration of hTR and telomerase activity after native gel electrophoresis of affinity-purified Raji cell extract (at 0.1 M NaCl). After clear native gel electrophoresis, one lane was stained with Coomassie blue (top panel; gel slice oriented from top to bottom as indicated), and one lane probed with a radiolabelled probe corresponding to hTR after transfer to nylon (middle panel). A third lane was sliced into 13 fractions, eluted into buffer and subjected to the telomere repeat amplification protocol, TRAP (bottom panel). (B) Relative mobility of telomerase after native gel electrophoresis and detection with a radiolabelled probe corresponding to hTR. Lane 1, in vitro transcribed hTR alone; Raji cell extracts purified at 0.1 M NaCl (low stringency; LS) and 0.6 M NaCl (high stringency; HS) at 4°C (lanes 2 and 3, respectively), 37°C (lanes 4 and 6) and after incubation with proteinase K (lanes 5 and 7). Brackets at right indicate position of gel slices. (C) The indicated treatments as in (B) were resolved by native gel electrophoresis, sliced into 13 fractions and subjected to TRAP (upper panels) and RT–PCR analysis to detect the native mobility of hTR (lower panels). Thyroglobulin (T) was loaded onto an adjacent lane of the native gel; its position is indicated with an arrow, top. Asterisk indicates TRAP internal PCR standard; arrows at right, hTR. The bottom panel represents PCR detection of hTR after native gel electrophoresis of purified, in vitro transcribed hTR alone (no added extract). At right, 1.0 and 0.2 µl of rabbit reticulocyte lysate (RRL) containing reconstituted telomerase activity, or water alone (−) as positive and negative controls for TRAP, respectively.

Mentions: In order to investigate the composition of endogenous telomerase, we employed a previously developed purification strategy using anti-sense affinity selection of hTR (19,22,23), with minor modifications (63). A biotinylated oligonucleotide containing a random sequence and a region of complementarity to the human telomerase RNA (hTR) (nt 50–64) was incubated with cell extracts from a human lymphoblastoma cell line (Raji). The oligonucleotide sequence differed slightly from that used in a previous study (nt 46–59) (19), and was based on optimization of the recovery of telomerase activity (63). Raji cells were chosen because of the relatively low level of hTR expression (35 copies/cell) (63) relative to other cell types (19,67,68), in order to maximize the ratio of affinity-purified hTR associated with telomerase activity. After incubation with Raji lysate, the affinity oligonucleotide was captured onto neutravidin beads, washed and eluted by virtue of a displacement oligonucleotide complementary to the affinity oligonucleotide (19,22,23,63). To further purify telomerase, this fraction was resolved on a clear, 3.5% w/v non-denaturing native acrylamide gel (Figure 1A). To locate active telomerase after electrophoresis, the native gel was separated into 1 cm slices numbered from top to bottom (e.g. Figure 1B, right), eluted into a buffered solution and subjected to the telomere repeat amplification protocol (TRAP) (Figure 1A, bottom panel). One adjacent lane of the native gel was stained with Coomassie blue dye, while another adjacent lane was probed for the presence of the telomerase RNA after transfer to nylon membrane (Figure 1A, top panels). These results demonstrated that the peak of endogenous telomerase activity and hTR co-migrated on the native gel at a position distinct from the bulk of proteins detected by Coomassie staining.Figure 1.


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 purification via affinity selection and native gel electrophoresis. (A) Co-migration of hTR and telomerase activity after native gel electrophoresis of affinity-purified Raji cell extract (at 0.1 M NaCl). After clear native gel electrophoresis, one lane was stained with Coomassie blue (top panel; gel slice oriented from top to bottom as indicated), and one lane probed with a radiolabelled probe corresponding to hTR after transfer to nylon (middle panel). A third lane was sliced into 13 fractions, eluted into buffer and subjected to the telomere repeat amplification protocol, TRAP (bottom panel). (B) Relative mobility of telomerase after native gel electrophoresis and detection with a radiolabelled probe corresponding to hTR. Lane 1, in vitro transcribed hTR alone; Raji cell extracts purified at 0.1 M NaCl (low stringency; LS) and 0.6 M NaCl (high stringency; HS) at 4°C (lanes 2 and 3, respectively), 37°C (lanes 4 and 6) and after incubation with proteinase K (lanes 5 and 7). Brackets at right indicate position of gel slices. (C) The indicated treatments as in (B) were resolved by native gel electrophoresis, sliced into 13 fractions and subjected to TRAP (upper panels) and RT–PCR analysis to detect the native mobility of hTR (lower panels). Thyroglobulin (T) was loaded onto an adjacent lane of the native gel; its position is indicated with an arrow, top. Asterisk indicates TRAP internal PCR standard; arrows at right, hTR. The bottom panel represents PCR detection of hTR after native gel electrophoresis of purified, in vitro transcribed hTR alone (no added extract). At right, 1.0 and 0.2 µl of rabbit reticulocyte lysate (RRL) containing reconstituted telomerase activity, or water alone (−) as positive and negative controls for TRAP, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr1243-F1: Telomerase purification via affinity selection and native gel electrophoresis. (A) Co-migration of hTR and telomerase activity after native gel electrophoresis of affinity-purified Raji cell extract (at 0.1 M NaCl). After clear native gel electrophoresis, one lane was stained with Coomassie blue (top panel; gel slice oriented from top to bottom as indicated), and one lane probed with a radiolabelled probe corresponding to hTR after transfer to nylon (middle panel). A third lane was sliced into 13 fractions, eluted into buffer and subjected to the telomere repeat amplification protocol, TRAP (bottom panel). (B) Relative mobility of telomerase after native gel electrophoresis and detection with a radiolabelled probe corresponding to hTR. Lane 1, in vitro transcribed hTR alone; Raji cell extracts purified at 0.1 M NaCl (low stringency; LS) and 0.6 M NaCl (high stringency; HS) at 4°C (lanes 2 and 3, respectively), 37°C (lanes 4 and 6) and after incubation with proteinase K (lanes 5 and 7). Brackets at right indicate position of gel slices. (C) The indicated treatments as in (B) were resolved by native gel electrophoresis, sliced into 13 fractions and subjected to TRAP (upper panels) and RT–PCR analysis to detect the native mobility of hTR (lower panels). Thyroglobulin (T) was loaded onto an adjacent lane of the native gel; its position is indicated with an arrow, top. Asterisk indicates TRAP internal PCR standard; arrows at right, hTR. The bottom panel represents PCR detection of hTR after native gel electrophoresis of purified, in vitro transcribed hTR alone (no added extract). At right, 1.0 and 0.2 µl of rabbit reticulocyte lysate (RRL) containing reconstituted telomerase activity, or water alone (−) as positive and negative controls for TRAP, respectively.
Mentions: In order to investigate the composition of endogenous telomerase, we employed a previously developed purification strategy using anti-sense affinity selection of hTR (19,22,23), with minor modifications (63). A biotinylated oligonucleotide containing a random sequence and a region of complementarity to the human telomerase RNA (hTR) (nt 50–64) was incubated with cell extracts from a human lymphoblastoma cell line (Raji). The oligonucleotide sequence differed slightly from that used in a previous study (nt 46–59) (19), and was based on optimization of the recovery of telomerase activity (63). Raji cells were chosen because of the relatively low level of hTR expression (35 copies/cell) (63) relative to other cell types (19,67,68), in order to maximize the ratio of affinity-purified hTR associated with telomerase activity. After incubation with Raji lysate, the affinity oligonucleotide was captured onto neutravidin beads, washed and eluted by virtue of a displacement oligonucleotide complementary to the affinity oligonucleotide (19,22,23,63). To further purify telomerase, this fraction was resolved on a clear, 3.5% w/v non-denaturing native acrylamide gel (Figure 1A). To locate active telomerase after electrophoresis, the native gel was separated into 1 cm slices numbered from top to bottom (e.g. Figure 1B, right), eluted into a buffered solution and subjected to the telomere repeat amplification protocol (TRAP) (Figure 1A, bottom panel). One adjacent lane of the native gel was stained with Coomassie blue dye, while another adjacent lane was probed for the presence of the telomerase RNA after transfer to nylon membrane (Figure 1A, top panels). These results demonstrated that the peak of endogenous telomerase activity and hTR co-migrated on the native gel at a position distinct from the bulk of proteins detected by Coomassie staining.Figure 1.

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