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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.


Expression of HA-tagged PinX1 truncations in HeLa cells. (A) Expression of HA-tagged PinX1 derivatives. HeLa cells transfected with PinX1 truncation mutants for 48 h were subjected to immunoblotting. β-actin was used as a loading control. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from three independent experiments. (C) Location of PCR primers in PinX1. Three pairs of PCR primers were indicated as a, b, and c. (D) RT-PCR showing levels of the HA-PinX1 derivatives. Total RNA was isolated from HeLa cells transfected with the indicated plasmids. RT-PCR was performed for PinX1 using the primers shown in C, indicated as a, b, and c. PinX1 and 18S rRNA were amplified for 20 and 18 cycles, respectively, and stained with ethidium bromide. (E) Quantification of relative PinX1 mRNA levels represented in D. PinX1 normalized to 18S rRNA was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from two independent experiments. (F) Fractionation of cell lysates. Cells collected at 2 days post-transfection were lysed by the freeze-thaw method, and fractions of supernatant and pellet were obtained after centrifugation of total cell lysates. A whole pellet resuspended in 10 μL of loading buffer and 40 μg of each of the total cell lysates and supernatants were loaded, followed by immunoblotting.
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f1-molce-38-9-814: Expression of HA-tagged PinX1 truncations in HeLa cells. (A) Expression of HA-tagged PinX1 derivatives. HeLa cells transfected with PinX1 truncation mutants for 48 h were subjected to immunoblotting. β-actin was used as a loading control. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from three independent experiments. (C) Location of PCR primers in PinX1. Three pairs of PCR primers were indicated as a, b, and c. (D) RT-PCR showing levels of the HA-PinX1 derivatives. Total RNA was isolated from HeLa cells transfected with the indicated plasmids. RT-PCR was performed for PinX1 using the primers shown in C, indicated as a, b, and c. PinX1 and 18S rRNA were amplified for 20 and 18 cycles, respectively, and stained with ethidium bromide. (E) Quantification of relative PinX1 mRNA levels represented in D. PinX1 normalized to 18S rRNA was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from two independent experiments. (F) Fractionation of cell lysates. Cells collected at 2 days post-transfection were lysed by the freeze-thaw method, and fractions of supernatant and pellet were obtained after centrifugation of total cell lysates. A whole pellet resuspended in 10 μL of loading buffer and 40 μg of each of the total cell lysates and supernatants were loaded, followed by immunoblotting.

Mentions: In the course of experimentation, we noticed that PinX1 truncation mutants were detected at different levels upon transfection. HA-tagged PinX1205–328, the C-terminal of PinX1, was relatively abundant compared to the N-terminal of PinX1, HA-PinX11–204. The level of HA-PinX11–204 was as low as 5% that of HA-PinX11–328, whereas HA-PinX1205–328 exhibited nearly six-fold higher level than HA-PinX11–328 (Figs. 1A and 1B). To test whether the differences in the levels of protein were derived by the efficiency of transcription, the levels of mRNA of the PinX1 truncations were determined by RT-PCR with three pairs of primers (Fig. 1C). PCR was performed with a condition that endogenous PinX1 remained undetected by using low number of cycles. Note that there is no PinX1 band in vector samples which exhibit 18S rRNA similar levels to other samples (Fig. 1D). The results showed no significant differences in the mRNA levels between HA-PinX11–328 and HA-PinX11-204, or between HA-PinX11–328 and HA-PinX1205–328, indicating that the efficiency of transcription was unlikely to contribute to the levels of the proteins (Figs. 1D and 1E). It was also possible that HA-PinX11–204 was aggregated in the pellet fraction during analysis, with only a small portion of the protein remaining in the supernatant fractions. To test this possibility, HA-PinX11–328 and HA-PinX11–204 were examined in total cell lysates as well as in both fractions of pellet and supernatant. The results showed low levels of HA-PinX11–204 in the total cell lysates, while insignificant amounts of the protein were present in the pellet fraction. Note that the whole fraction was loaded for the pellet while only a portion was loaded for the supernatants (Fig. 1F). Taken together, this indicated that the PinX1 truncations were accumulated at different levels, and particularly, that the low levels of the N-terminal of PinX1 were not to be due to weak transcriptional activity or aggregation of the protein in the pellet fraction.


Increased Stability of Nucleolar PinX1 in the Presence of TERT.

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

Expression of HA-tagged PinX1 truncations in HeLa cells. (A) Expression of HA-tagged PinX1 derivatives. HeLa cells transfected with PinX1 truncation mutants for 48 h were subjected to immunoblotting. β-actin was used as a loading control. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from three independent experiments. (C) Location of PCR primers in PinX1. Three pairs of PCR primers were indicated as a, b, and c. (D) RT-PCR showing levels of the HA-PinX1 derivatives. Total RNA was isolated from HeLa cells transfected with the indicated plasmids. RT-PCR was performed for PinX1 using the primers shown in C, indicated as a, b, and c. PinX1 and 18S rRNA were amplified for 20 and 18 cycles, respectively, and stained with ethidium bromide. (E) Quantification of relative PinX1 mRNA levels represented in D. PinX1 normalized to 18S rRNA was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from two independent experiments. (F) Fractionation of cell lysates. Cells collected at 2 days post-transfection were lysed by the freeze-thaw method, and fractions of supernatant and pellet were obtained after centrifugation of total cell lysates. A whole pellet resuspended in 10 μL of loading buffer and 40 μg of each of the total cell lysates and supernatants were loaded, followed by immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588725&req=5

f1-molce-38-9-814: Expression of HA-tagged PinX1 truncations in HeLa cells. (A) Expression of HA-tagged PinX1 derivatives. HeLa cells transfected with PinX1 truncation mutants for 48 h were subjected to immunoblotting. β-actin was used as a loading control. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from three independent experiments. (C) Location of PCR primers in PinX1. Three pairs of PCR primers were indicated as a, b, and c. (D) RT-PCR showing levels of the HA-PinX1 derivatives. Total RNA was isolated from HeLa cells transfected with the indicated plasmids. RT-PCR was performed for PinX1 using the primers shown in C, indicated as a, b, and c. PinX1 and 18S rRNA were amplified for 20 and 18 cycles, respectively, and stained with ethidium bromide. (E) Quantification of relative PinX1 mRNA levels represented in D. PinX1 normalized to 18S rRNA was quantified as the ratio relative to HA-PinX11–328. Error bars were derived from two independent experiments. (F) Fractionation of cell lysates. Cells collected at 2 days post-transfection were lysed by the freeze-thaw method, and fractions of supernatant and pellet were obtained after centrifugation of total cell lysates. A whole pellet resuspended in 10 μL of loading buffer and 40 μg of each of the total cell lysates and supernatants were loaded, followed by immunoblotting.
Mentions: In the course of experimentation, we noticed that PinX1 truncation mutants were detected at different levels upon transfection. HA-tagged PinX1205–328, the C-terminal of PinX1, was relatively abundant compared to the N-terminal of PinX1, HA-PinX11–204. The level of HA-PinX11–204 was as low as 5% that of HA-PinX11–328, whereas HA-PinX1205–328 exhibited nearly six-fold higher level than HA-PinX11–328 (Figs. 1A and 1B). To test whether the differences in the levels of protein were derived by the efficiency of transcription, the levels of mRNA of the PinX1 truncations were determined by RT-PCR with three pairs of primers (Fig. 1C). PCR was performed with a condition that endogenous PinX1 remained undetected by using low number of cycles. Note that there is no PinX1 band in vector samples which exhibit 18S rRNA similar levels to other samples (Fig. 1D). The results showed no significant differences in the mRNA levels between HA-PinX11–328 and HA-PinX11-204, or between HA-PinX11–328 and HA-PinX1205–328, indicating that the efficiency of transcription was unlikely to contribute to the levels of the proteins (Figs. 1D and 1E). It was also possible that HA-PinX11–204 was aggregated in the pellet fraction during analysis, with only a small portion of the protein remaining in the supernatant fractions. To test this possibility, HA-PinX11–328 and HA-PinX11–204 were examined in total cell lysates as well as in both fractions of pellet and supernatant. The results showed low levels of HA-PinX11–204 in the total cell lysates, while insignificant amounts of the protein were present in the pellet fraction. Note that the whole fraction was loaded for the pellet while only a portion was loaded for the supernatants (Fig. 1F). Taken together, this indicated that the PinX1 truncations were accumulated at different levels, and particularly, that the low levels of the N-terminal of PinX1 were not to be due to weak transcriptional activity or aggregation of the protein in the pellet fraction.

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.