Limits...
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.


POT1a associates with CTC1 and STN1 in vitro.(A) In vitro co-immunoprecipitation (co-IP) results for RRL-expressed T7-tagged POT1a interactions with labeled CTC1ΔN, STN1, and TEN1. Negative control (beads conjugated with T7-tag antibody) was performed without tagged POT1a. (I) denotes protein input, (B) indicates bound protein. (B) Co-IP results for RRL-expressed T7 tagged TEN1 with labeled POT1a (P; lane 6), STN1 (S; lane 4) or both proteins (“PS”, lane 2). The beads control contained no T7 tagged TEN1 (lane 8). (C) In vitro Co-IP competition assay using E. coli-expressed TEN1 and POT1a OB1 detected by coomassie stain, and RRL-expressed 35S methionine labeled STN1 detected by autoradiography. Protein inputs are shown in lanes 1–3. Bracket adjacent to lane 1 denotes non-specific RRL proteins in the STN1 expression reaction (lane 1, top). TEN1 was incubated with STN1 and increasing concentrations of POT1a OB1 (lanes 8–10). 50× BSA was used as a control (lane 7) IP of POT1a was performed independently to verify its interaction with STN1 (lane 5). Beads alone was used to monitor background binding of STN1 protein (lane 4).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199523&req=5

pgen-1004738-g005: POT1a associates with CTC1 and STN1 in vitro.(A) In vitro co-immunoprecipitation (co-IP) results for RRL-expressed T7-tagged POT1a interactions with labeled CTC1ΔN, STN1, and TEN1. Negative control (beads conjugated with T7-tag antibody) was performed without tagged POT1a. (I) denotes protein input, (B) indicates bound protein. (B) Co-IP results for RRL-expressed T7 tagged TEN1 with labeled POT1a (P; lane 6), STN1 (S; lane 4) or both proteins (“PS”, lane 2). The beads control contained no T7 tagged TEN1 (lane 8). (C) In vitro Co-IP competition assay using E. coli-expressed TEN1 and POT1a OB1 detected by coomassie stain, and RRL-expressed 35S methionine labeled STN1 detected by autoradiography. Protein inputs are shown in lanes 1–3. Bracket adjacent to lane 1 denotes non-specific RRL proteins in the STN1 expression reaction (lane 1, top). TEN1 was incubated with STN1 and increasing concentrations of POT1a OB1 (lanes 8–10). 50× BSA was used as a control (lane 7) IP of POT1a was performed independently to verify its interaction with STN1 (lane 5). Beads alone was used to monitor background binding of STN1 protein (lane 4).

Mentions: Recent studies show that human POT1 and mouse POT1b bind CTC1 and STN1 [46], [47], [64]. Additional contacts between TPP1 and CTC1 and TPP1 and STN1 have been observed [46], [64], [65]. Therefore, we asked if POT1a binds individual CST subunits in vitro via co-immunoprecipitation assays using rabbit reticulocyte lysate (RRL) expressed proteins. We were unable to express intact full length CTC1, and so we employed an amino-terminal deletion construct (CTC1ΔN) that was sufficient to bind STN1 and the DNA Pol α subunit, ICU2 [4], [39]. POT1a was tagged with T7 on its amino terminus and immunoprecipitation (IP) was performed using T7 antibody-conjugated agarose beads. Binding was assessed by the ability of POT1a to co-precipitate 35S-methionine labeled CTC1ΔN, STN1, or TEN1. We detected POT1a binding to CTC1ΔN and STN1, but no interaction between TEN1 and POT1a was observed (Fig. 5A).


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 associates with CTC1 and STN1 in vitro.(A) In vitro co-immunoprecipitation (co-IP) results for RRL-expressed T7-tagged POT1a interactions with labeled CTC1ΔN, STN1, and TEN1. Negative control (beads conjugated with T7-tag antibody) was performed without tagged POT1a. (I) denotes protein input, (B) indicates bound protein. (B) Co-IP results for RRL-expressed T7 tagged TEN1 with labeled POT1a (P; lane 6), STN1 (S; lane 4) or both proteins (“PS”, lane 2). The beads control contained no T7 tagged TEN1 (lane 8). (C) In vitro Co-IP competition assay using E. coli-expressed TEN1 and POT1a OB1 detected by coomassie stain, and RRL-expressed 35S methionine labeled STN1 detected by autoradiography. Protein inputs are shown in lanes 1–3. Bracket adjacent to lane 1 denotes non-specific RRL proteins in the STN1 expression reaction (lane 1, top). TEN1 was incubated with STN1 and increasing concentrations of POT1a OB1 (lanes 8–10). 50× BSA was used as a control (lane 7) IP of POT1a was performed independently to verify its interaction with STN1 (lane 5). Beads alone was used to monitor background binding of STN1 protein (lane 4).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004738-g005: POT1a associates with CTC1 and STN1 in vitro.(A) In vitro co-immunoprecipitation (co-IP) results for RRL-expressed T7-tagged POT1a interactions with labeled CTC1ΔN, STN1, and TEN1. Negative control (beads conjugated with T7-tag antibody) was performed without tagged POT1a. (I) denotes protein input, (B) indicates bound protein. (B) Co-IP results for RRL-expressed T7 tagged TEN1 with labeled POT1a (P; lane 6), STN1 (S; lane 4) or both proteins (“PS”, lane 2). The beads control contained no T7 tagged TEN1 (lane 8). (C) In vitro Co-IP competition assay using E. coli-expressed TEN1 and POT1a OB1 detected by coomassie stain, and RRL-expressed 35S methionine labeled STN1 detected by autoradiography. Protein inputs are shown in lanes 1–3. Bracket adjacent to lane 1 denotes non-specific RRL proteins in the STN1 expression reaction (lane 1, top). TEN1 was incubated with STN1 and increasing concentrations of POT1a OB1 (lanes 8–10). 50× BSA was used as a control (lane 7) IP of POT1a was performed independently to verify its interaction with STN1 (lane 5). Beads alone was used to monitor background binding of STN1 protein (lane 4).
Mentions: Recent studies show that human POT1 and mouse POT1b bind CTC1 and STN1 [46], [47], [64]. Additional contacts between TPP1 and CTC1 and TPP1 and STN1 have been observed [46], [64], [65]. Therefore, we asked if POT1a binds individual CST subunits in vitro via co-immunoprecipitation assays using rabbit reticulocyte lysate (RRL) expressed proteins. We were unable to express intact full length CTC1, and so we employed an amino-terminal deletion construct (CTC1ΔN) that was sufficient to bind STN1 and the DNA Pol α subunit, ICU2 [4], [39]. POT1a was tagged with T7 on its amino terminus and immunoprecipitation (IP) was performed using T7 antibody-conjugated agarose beads. Binding was assessed by the ability of POT1a to co-precipitate 35S-methionine labeled CTC1ΔN, STN1, or TEN1. We detected POT1a binding to CTC1ΔN and STN1, but no interaction between TEN1 and POT1a was observed (Fig. 5A).

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.