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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

POT1a stimulates telomerase activity of the TER1 RNP.(A) TP-TRAP analysis from two independent biological replicates wild type, pot1a, ter2, and pot1a ter2 mutants. (B) Results of quantitative TRAP (qTRAP). Error bars represent standard error of the mean from three biological replicates.
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pgen-1004738-g003: POT1a stimulates telomerase activity of the TER1 RNP.(A) TP-TRAP analysis from two independent biological replicates wild type, pot1a, ter2, and pot1a ter2 mutants. (B) Results of quantitative TRAP (qTRAP). Error bars represent standard error of the mean from three biological replicates.

Mentions: To determine if the decreased telomerase activity associated with pot1a mutants is specific to the TER1 RNP complex, we performed TP-TRAP on ter2 seedling extracts. The product profiles were nearly identical to wild type (Fig. 3A), indicating the TER1 RNP efficiently synthesizes telomeric DNA in wild type plants. We confirmed that POT1a modulates the TER1 RNP by analyzing pot1a ter2 mutants. Long products were reduced in the double mutants, but not to the same extent as pot1a (Fig. 3A). In agreement with previous results showing that TER2 negatively regulates TER1 RNP [54], quantitative TRAP (qTRAP) revealed a higher level of telomerase activity in ter2 mutants relative to wild type (Fig. 3B), which could explain why the TP-TRAP and qTRAP signal is higher in pot1a ter2 than pot1a (Fig. 3A and B). Since the TER1 RNP is the only functional telomerase complex in pot1a ter2 mutants, the data indicate POT1a distinctly modulates this complex.


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)

POT1a stimulates telomerase activity of the TER1 RNP.(A) TP-TRAP analysis from two independent biological replicates wild type, pot1a, ter2, and pot1a ter2 mutants. (B) Results of quantitative TRAP (qTRAP). Error bars represent standard error of the mean from three biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004738-g003: POT1a stimulates telomerase activity of the TER1 RNP.(A) TP-TRAP analysis from two independent biological replicates wild type, pot1a, ter2, and pot1a ter2 mutants. (B) Results of quantitative TRAP (qTRAP). Error bars represent standard error of the mean from three biological replicates.
Mentions: To determine if the decreased telomerase activity associated with pot1a mutants is specific to the TER1 RNP complex, we performed TP-TRAP on ter2 seedling extracts. The product profiles were nearly identical to wild type (Fig. 3A), indicating the TER1 RNP efficiently synthesizes telomeric DNA in wild type plants. We confirmed that POT1a modulates the TER1 RNP by analyzing pot1a ter2 mutants. Long products were reduced in the double mutants, but not to the same extent as pot1a (Fig. 3A). In agreement with previous results showing that TER2 negatively regulates TER1 RNP [54], quantitative TRAP (qTRAP) revealed a higher level of telomerase activity in ter2 mutants relative to wild type (Fig. 3B), which could explain why the TP-TRAP and qTRAP signal is higher in pot1a ter2 than pot1a (Fig. 3A and B). Since the TER1 RNP is the only functional telomerase complex in pot1a ter2 mutants, the data indicate POT1a distinctly modulates this complex.

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