Identification of human TERT elements necessary for telomerase recruitment to telomeres
- Howard Hughes Medical Institute, University of Colorado Boulder, United States
Figures
Figure 1 with 2 supplements
Identification of mutants in the TEN-domain of hTERT that affect the interaction with POT1-TPP1 in vitro but not enzymatic activity.
(A) Domain structures of hTERT and TPP1 proteins. Question mark, proposed interaction between the TEL-patch on TPP1 and the N-DAT region of the TEN-domain of hTERT. In TPP1, the OB-domain (OB) is …
Figure 1—figure supplement 1
Additional TEN-domain mutants tested in this study.
(A) Direct telomerase assays of additional hTERT TEN-domain mutants (all with WT hTR) without (−) and with (+) WT POT1-TPP1. LC and +4 marker as in Figure 1E. Quantification in Table 1. (B) Western …
Figure 1—figure supplement 2
Comparison of methods for quantifying RAP stimulation by POT1-TPP1.
(A) Example of linear regression of data from one replicate that was used to calculate RAP stimulation by POT1-TPP1 in Figure 1H, using the decay method. All four assays included wild-type …
Figure 2 with 1 supplement
Conserved basic residues K78 and R132 are found in close proximity on the surface of the TEN-domain.
Cartoon representation of the TEN-domain crystal structure from Tetrahymena thermophila (PDB: 2B2A). Amino acid numbers are from Tetrahymena (human counterparts in parentheses). Anchor site (DNA …
Figure 2—figure supplement 1
Alignment and secondary structure prediction for human TEN-domain.
Secondary structure prediction for hTERT TEN domain and alignment to selected eukaryotic TERT TEN domains. Human secondary structure prediction made with JPRED3 (Cole et al., 2008), while T. …
Figure 3
TEN-domain mutations disrupt telomere localization of telomerase.
(A) Western blots of lysates of HeLa cells transfected with expression plasmids for various hTERT alleles and hTR, probed with an antibody against hTERT. Actin was used as a loading control. (B) …
Figure 4
TEN-domain mutants that do not localize to telomeres fail to elongate telomeres in vivo.
(A) Western blot probed for hTERT and for Actin as a loading control, showing hTERT expression in lysates of parental HeLa cells and cell lines stably expressing mCherry-hTERT variants. hTR was not …
Figure 5 with 3 supplements
A compensatory mutation in the TEL-patch of TPP1 rescues RAP stimulation of TEN-domain mutant telomerase in vitro.
(A) Schematic of the charge-swap experiment to test the interaction between specific amino acids on the TEL-patch on TPP1 and the N-DAT region of the TEN-domain of hTERT. Predicted experimental …
Figure 5—figure supplement 1
The charge-swap is statistically significant by alternative quantitation methods.
RAP stimulation by PT calculated using the fraction method to quantitate data presented in Figure 5D (n = 3, Mean ± SD, *p < 0.05, **p < 0.01, Student's t test).
Figure 5—figure supplement 2
E215K TPP1 rescues RAP stimulation of K78E mutant telomerase produced in RRLs.
(A) Western-blot for hTERT produced in RRLs. Also shown is a serial dilution of overexpressed telomerase immuno-purified from HEK293T cells. 1X signifies amount of telomerase used in direct …
Figure 5—figure supplement 3
The IPF allele V144M is deficient in RAP stimulation by TPP1.
(A) Direct telomerase assays of wild type and V144M hTERT telomerases in the presence of WT, E169K, or E215K POT1-TPP1. LC and +4 marker as in Figure 1E. (B) Western blot of the relative quantities …
Figure 6 with 1 supplement
A compensatory mutation in the TEL-patch of TPP1 rescues TEN-domain mutant telomerase binding to the TPP1 OB-domain in vivo.
(A) Model showing the experimental design. Fusion of the OB-domain of TPP1 to the lac repressor (LacI) recruits the OB-domain to a single non-telomeric chromosomal locus (LacO array), allowing the …
Figure 6—figure supplement 1
Additional examples of U2OS 2-6-3 cells expressing GFP-TPP1 OB-LacI and mCherry-telomerase.
Fluorescence images showing the localization of GFP-TPP1-OB-LacI and mCherry-hTERT fusion proteins in cell nuclei stained by DAPI. Cells were fixed, permeabilized, and stained with DAPI. The …
Figure 7
TPP1 E215K rescues telomere maintenance in cells expressing hTERT K78E.
(A) Western blot probed for TPP1-FLAG and for Actin as a loading control, showing TPP1 expression in lysates of parental HeLa cells and cell lines stably expressing TPP1-FLAG and mCherry-hTERT …
Figure 8
A model for the recruitment of telomerase to telomeres through the direct interaction between the TEL-patch of TPP1 and the hTERT TEN-domain.
Throughout the cell cycle, telomerase associates with TCAB1 and localizes to Cajal bodies. During late S/G2, telomerase is recruited to telomeres through a direct interaction between K78 on the …
Tables
Table 1
Telomerase TEN domain mutant activity, processivity, and RAP stimulation by wild-type TPP1
| TEN domain mutant | Activity % of WT* | Processivity % of WT | RAP stimulation by PT† % of WT |
|---|---|---|---|
| R72E | 48 ± 3 | 99 ± 1 | 91 ± 2 |
| K78A | 19 ± 2 | 101 ± 7 | 88 ± 2 |
| K78E | 92 ± 1 | 100 ± 1 | 68 ± 5 |
| R87E;R91E;K94E | 74 ± 6 | 75 ± 0.3 | 61 ± 4 |
| R120E | 99 | 96 | 55 |
| K78E;R120E | 82 ± 18 | 87 ± 3 | 38 ± 2 |
| R132E | 73 ± 2 | 92 ± 3 | 24 ± 2 |
| R132E;K78E | 63 ± 10 | 94 ± 8 | 18 ± 1 |
| R142E | 97 ± 7 | 111 ± 10 | 103 ± 4 |
| R143E | 16 ± 12 | 81 ± 3 | N.D.‡ |
| R142E;R143E | 4 ± 1 | N.D.‡ | N.D.‡ |
| V144M | 48 ± 6 | 95 ± 5 | 44 ± 7 |
-
*
Percentage of wild-type telomerase activity, processivity, or RAP stimulation. Activity values normalized to hTERT levels, loading control, ± standard deviation for 2 or more replicates.
-
†
Repeat addition processivity (RAP) stimulation upon addition of WT POT1-TPP1, values relative to WT telomerase with WT POT1-TPP1 (i.e. RAP stimulation by PT % of WT = ((WT PT RAP stimulation of telomerase mutant)/(WT PT RAP stimulation WT telomerase))*100).
-
‡
Not determined (N.D.) due to low telomerase activity.
