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Increased Stability of Nucleolar PinX1 in the Presence of TERT.

Keo P, Choi JS, Bae J, Shim YH, Oh BK - Mol. Cells (2015)

Bottom Line: Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells.However, PinX1(1-204) was degraded regardless of the TERT status.These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea.

ABSTRACT
PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1(205-328), a nucleolar fragment, was much more stable than the N-terminal of PinX1(1-204), a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX1(1-328) and PinX1(205-328), nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX1(1-204) was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

No MeSH data available.


TERT positively regulates the stability of PinX1. (A) PinX1 protein is less stable in TERT-depleted cells. HeLa cells transfected with siTERT for 48 h were treated with cycloheximide (CHX) for the indicated times, followed by immunoblot. (B) Quantification of PinX1 represented in A. PinX1 level normalized to β-actin was quantified relative to the level at 0 h. (C) qRT-PCR for TERT in the samples shown in A. Total RNA isolated from a portion of the cells tested in A was subjected to qRT-PCR. TERT mRNA was quantified using the 2−ΔΔCT method, for which β-actin was determined as an internal control. TERT mRNA detected in si-Control-treated cells at 0 h was defined as 1.0. (D) PinX1 protein is more stable in TERT-overexpressing cells. HeLa cells transfected with TERT-myc or vector for 48 h were treated with cycloheximide for the indicated times, and lysates were prepared for immunoblot. PinX1, TERT-myc, and β-actin were detected. (E) Quantification of PinX1 represented in D. PinX1 level was quantified as described in B. Error bars shown in graphs B and C represent the S.D. of the mean from three independent experiments and those in E from five independent assays.
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f4-molce-38-9-814: TERT positively regulates the stability of PinX1. (A) PinX1 protein is less stable in TERT-depleted cells. HeLa cells transfected with siTERT for 48 h were treated with cycloheximide (CHX) for the indicated times, followed by immunoblot. (B) Quantification of PinX1 represented in A. PinX1 level normalized to β-actin was quantified relative to the level at 0 h. (C) qRT-PCR for TERT in the samples shown in A. Total RNA isolated from a portion of the cells tested in A was subjected to qRT-PCR. TERT mRNA was quantified using the 2−ΔΔCT method, for which β-actin was determined as an internal control. TERT mRNA detected in si-Control-treated cells at 0 h was defined as 1.0. (D) PinX1 protein is more stable in TERT-overexpressing cells. HeLa cells transfected with TERT-myc or vector for 48 h were treated with cycloheximide for the indicated times, and lysates were prepared for immunoblot. PinX1, TERT-myc, and β-actin were detected. (E) Quantification of PinX1 represented in D. PinX1 level was quantified as described in B. Error bars shown in graphs B and C represent the S.D. of the mean from three independent experiments and those in E from five independent assays.

Mentions: In a previous study, we found GFP-PinX1 to be less abundant in TERT-depleted cells, which were generated by treatment with siRNA targeting TERT, named siTERT (Yoo et al., 2014). To explore the functional connection between TERT and PinX1, endogenous PinX1 protein was measured in siTERT-treated HeLa cells (Fig. 3A). Cells transfected with siTERT for 48 h, which showed no noticeable changes on cell growth, were subjected to immunoblotting and qRT-PCR. Immunoblotting revealed that the level of PinX1 protein was slightly decreased in siTERT-treated cells compared to the control cells although the effect was mild (0.87 ± 0.143, n = 9, p = 0.027) (Fig. 3A). PinX1 mRNA was also measured by qRT-PCR to ensure that there was no off-target effect of siTERT (Fig. 3B), and TERT status was verified by qRT-PCR (Fig. 3B). Next, the level of PinX1 protein was measured in cells transfected with TERT-myc. Expression of TERT-myc was verified by immunoblotting (Fig. 3C) and/or qRT-PCR (1 ×103 − 1 × 104 of TERT mRNA in TERT-myc-transfected cells versus 1.0 in the vector cells, data not shown). Immunoblotting showed that the level of PinX1 rather varied among the assays (Fig. 3C). We further examined the level of PinX1 protein in the ALT cell line GM847 and in stable TERT-expressing GM847 cells (Jung et al., 2013). Interestingly, PinX1 protein was more abundant in the TERT-expressing GM847 cells (1.5 ± 0.07) than in the TERT-negative GM847 cells (Fig. 3D). The increase in PinX1 accumulation in response to excess TERT presented in Fig. 3D are more convincing probably because the amplitude of the variations in TERT levels are larger. Based on these results, it was tempting to speculate that there might be a role of TERT on PinX1 accumulation. To address this possibility, we investigated whether the stability of PinX1 protein is affected by TERT. A PinX1 stability assay was performed in TERT-depleted and -overexpressing cells. The assays showed that PinX1 was degraded more rapidly in the siTERT-treated cells compared to the siControl-treated cells upon treatment with CHX (Figs. 4A and 4B). Depletion of TERT in the samples was verified by qRT-PCR (Fig. 4C). In contrast, PinX1 was more stably maintained in the TERT-myc-expressing cells compared to vector-transfected cells (Figs. 4D and 4E). Again, amplification of TERT in the samples was verified by immunoblotting (Fig. 4D) and/or qRT-PCR (data not shown). Our findings reveal that the stability of PinX1 is maintained in the presence of TERT.


Increased Stability of Nucleolar PinX1 in the Presence of TERT.

Keo P, Choi JS, Bae J, Shim YH, Oh BK - Mol. Cells (2015)

TERT positively regulates the stability of PinX1. (A) PinX1 protein is less stable in TERT-depleted cells. HeLa cells transfected with siTERT for 48 h were treated with cycloheximide (CHX) for the indicated times, followed by immunoblot. (B) Quantification of PinX1 represented in A. PinX1 level normalized to β-actin was quantified relative to the level at 0 h. (C) qRT-PCR for TERT in the samples shown in A. Total RNA isolated from a portion of the cells tested in A was subjected to qRT-PCR. TERT mRNA was quantified using the 2−ΔΔCT method, for which β-actin was determined as an internal control. TERT mRNA detected in si-Control-treated cells at 0 h was defined as 1.0. (D) PinX1 protein is more stable in TERT-overexpressing cells. HeLa cells transfected with TERT-myc or vector for 48 h were treated with cycloheximide for the indicated times, and lysates were prepared for immunoblot. PinX1, TERT-myc, and β-actin were detected. (E) Quantification of PinX1 represented in D. PinX1 level was quantified as described in B. Error bars shown in graphs B and C represent the S.D. of the mean from three independent experiments and those in E from five independent assays.
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f4-molce-38-9-814: TERT positively regulates the stability of PinX1. (A) PinX1 protein is less stable in TERT-depleted cells. HeLa cells transfected with siTERT for 48 h were treated with cycloheximide (CHX) for the indicated times, followed by immunoblot. (B) Quantification of PinX1 represented in A. PinX1 level normalized to β-actin was quantified relative to the level at 0 h. (C) qRT-PCR for TERT in the samples shown in A. Total RNA isolated from a portion of the cells tested in A was subjected to qRT-PCR. TERT mRNA was quantified using the 2−ΔΔCT method, for which β-actin was determined as an internal control. TERT mRNA detected in si-Control-treated cells at 0 h was defined as 1.0. (D) PinX1 protein is more stable in TERT-overexpressing cells. HeLa cells transfected with TERT-myc or vector for 48 h were treated with cycloheximide for the indicated times, and lysates were prepared for immunoblot. PinX1, TERT-myc, and β-actin were detected. (E) Quantification of PinX1 represented in D. PinX1 level was quantified as described in B. Error bars shown in graphs B and C represent the S.D. of the mean from three independent experiments and those in E from five independent assays.
Mentions: In a previous study, we found GFP-PinX1 to be less abundant in TERT-depleted cells, which were generated by treatment with siRNA targeting TERT, named siTERT (Yoo et al., 2014). To explore the functional connection between TERT and PinX1, endogenous PinX1 protein was measured in siTERT-treated HeLa cells (Fig. 3A). Cells transfected with siTERT for 48 h, which showed no noticeable changes on cell growth, were subjected to immunoblotting and qRT-PCR. Immunoblotting revealed that the level of PinX1 protein was slightly decreased in siTERT-treated cells compared to the control cells although the effect was mild (0.87 ± 0.143, n = 9, p = 0.027) (Fig. 3A). PinX1 mRNA was also measured by qRT-PCR to ensure that there was no off-target effect of siTERT (Fig. 3B), and TERT status was verified by qRT-PCR (Fig. 3B). Next, the level of PinX1 protein was measured in cells transfected with TERT-myc. Expression of TERT-myc was verified by immunoblotting (Fig. 3C) and/or qRT-PCR (1 ×103 − 1 × 104 of TERT mRNA in TERT-myc-transfected cells versus 1.0 in the vector cells, data not shown). Immunoblotting showed that the level of PinX1 rather varied among the assays (Fig. 3C). We further examined the level of PinX1 protein in the ALT cell line GM847 and in stable TERT-expressing GM847 cells (Jung et al., 2013). Interestingly, PinX1 protein was more abundant in the TERT-expressing GM847 cells (1.5 ± 0.07) than in the TERT-negative GM847 cells (Fig. 3D). The increase in PinX1 accumulation in response to excess TERT presented in Fig. 3D are more convincing probably because the amplitude of the variations in TERT levels are larger. Based on these results, it was tempting to speculate that there might be a role of TERT on PinX1 accumulation. To address this possibility, we investigated whether the stability of PinX1 protein is affected by TERT. A PinX1 stability assay was performed in TERT-depleted and -overexpressing cells. The assays showed that PinX1 was degraded more rapidly in the siTERT-treated cells compared to the siControl-treated cells upon treatment with CHX (Figs. 4A and 4B). Depletion of TERT in the samples was verified by qRT-PCR (Fig. 4C). In contrast, PinX1 was more stably maintained in the TERT-myc-expressing cells compared to vector-transfected cells (Figs. 4D and 4E). Again, amplification of TERT in the samples was verified by immunoblotting (Fig. 4D) and/or qRT-PCR (data not shown). Our findings reveal that the stability of PinX1 is maintained in the presence of TERT.

Bottom Line: Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells.However, PinX1(1-204) was degraded regardless of the TERT status.These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea.

ABSTRACT
PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1(205-328), a nucleolar fragment, was much more stable than the N-terminal of PinX1(1-204), a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX1(1-328) and PinX1(205-328), nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX1(1-204) was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

No MeSH data available.