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POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.

Renfrew KB, Song X, Lee JR, Arora A, Shippen DE - PLoS Genet. (2014)

Bottom Line: We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo.Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity.Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Protection of Telomeres 1 (POT1) is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1) telomere complex. Analysis of pot1a mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

No MeSH data available.


Related in: MedlinePlus

STN1, but not TEN1 associates with enzymatically active telomerase in vivo.(A) Protein extract from wild type or pot1a seedlings was used for immunoprecipitation with STN1 or TERT antibody. IP samples and extract input were subjected to conventional TRAP (A) or western blot (B) analysis with STN1 antibody. (C) Protein extract from wild type seedlings was used for IP with TEN1 antibody. IP samples and extract input were subjected to conventional TRAP or (D) western blot to monitor for TEN1 protein after IP.
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pgen-1004738-g006: STN1, but not TEN1 associates with enzymatically active telomerase in vivo.(A) Protein extract from wild type or pot1a seedlings was used for immunoprecipitation with STN1 or TERT antibody. IP samples and extract input were subjected to conventional TRAP (A) or western blot (B) analysis with STN1 antibody. (C) Protein extract from wild type seedlings was used for IP with TEN1 antibody. IP samples and extract input were subjected to conventional TRAP or (D) western blot to monitor for TEN1 protein after IP.

Mentions: The discovery of in vitro interactions between POT1a with STN1 and CTC1 raised the possibility that these CST components associate with enzymatically active telomerase in vivo (Fig. 6). To test this idea, we generated a STN1 antibody that could be used for IP-TRAP. Western blot analysis confirmed that the antibody specifically recognizes STN1 (Fig. 6B). IP-TRAP using TERT antibody as a control revealed abundant telomerase activity (Fig. 6A). Strikingly, IP-TRAP with STN1 antibody gave a similar result. Western blot analysis verified that STN1 was precipitated in the reaction (Fig. 6B). Telomerase activity was not detected in an IP with pre-immune sera and was removed by RNaseA treatment, indicating that the STN1 interaction with telomerase was specific. Importantly, STN1 protein was present in the TERT IP (Fig. 6B), confirming the association of these molecules in vivo. IP of a transgenic CTC1-CFP protein also pulled down active telomerase as well as POT1a (Fig. S6). These findings indicate that both STN1 and CTC1 are associated with enzymatically active telomerase in vivo.


POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.

Renfrew KB, Song X, Lee JR, Arora A, Shippen DE - PLoS Genet. (2014)

STN1, but not TEN1 associates with enzymatically active telomerase in vivo.(A) Protein extract from wild type or pot1a seedlings was used for immunoprecipitation with STN1 or TERT antibody. IP samples and extract input were subjected to conventional TRAP (A) or western blot (B) analysis with STN1 antibody. (C) Protein extract from wild type seedlings was used for IP with TEN1 antibody. IP samples and extract input were subjected to conventional TRAP or (D) western blot to monitor for TEN1 protein after IP.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199523&req=5

pgen-1004738-g006: STN1, but not TEN1 associates with enzymatically active telomerase in vivo.(A) Protein extract from wild type or pot1a seedlings was used for immunoprecipitation with STN1 or TERT antibody. IP samples and extract input were subjected to conventional TRAP (A) or western blot (B) analysis with STN1 antibody. (C) Protein extract from wild type seedlings was used for IP with TEN1 antibody. IP samples and extract input were subjected to conventional TRAP or (D) western blot to monitor for TEN1 protein after IP.
Mentions: The discovery of in vitro interactions between POT1a with STN1 and CTC1 raised the possibility that these CST components associate with enzymatically active telomerase in vivo (Fig. 6). To test this idea, we generated a STN1 antibody that could be used for IP-TRAP. Western blot analysis confirmed that the antibody specifically recognizes STN1 (Fig. 6B). IP-TRAP using TERT antibody as a control revealed abundant telomerase activity (Fig. 6A). Strikingly, IP-TRAP with STN1 antibody gave a similar result. Western blot analysis verified that STN1 was precipitated in the reaction (Fig. 6B). Telomerase activity was not detected in an IP with pre-immune sera and was removed by RNaseA treatment, indicating that the STN1 interaction with telomerase was specific. Importantly, STN1 protein was present in the TERT IP (Fig. 6B), confirming the association of these molecules in vivo. IP of a transgenic CTC1-CFP protein also pulled down active telomerase as well as POT1a (Fig. S6). These findings indicate that both STN1 and CTC1 are associated with enzymatically active telomerase in vivo.

Bottom Line: We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo.Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity.Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Protection of Telomeres 1 (POT1) is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1) telomere complex. Analysis of pot1a mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

No MeSH data available.


Related in: MedlinePlus