Volume 27, Issue 6, Pages 882-889 (September 2007) Human Telomerase RNA Accumulation in Cajal Bodies Facilitates Telomerase Recruitment to Telomeres and Telomere Elongation  Gaël Cristofari, Emem Adolf, Patrick Reichenbach, Katarzyna Sikora, Rebecca M. Terns, Michael P. Terns, Joachim Lingner  Molecular Cell  Volume 27, Issue 6, Pages 882-889 (September 2007) DOI: 10.1016/j.molcel.2007.07.020 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Telomerase RNA with Mutation in the CAB Box Accumulates Normally When Transiently Expressed in HEK293T Cells (A) Schematic representation of hTR secondary structure showing the location of the CAB box point mutations used in this study (underlined). The effect of these mutations on hTR subnuclear localization is indicated (as previously described (Jady et al., 2004) and as also shown in Figure S1). (B–D) HEK293T cells were transiently cotransfected with hTR- and TAP-hTERT-expressing plasmids. Extracts were prepared 48 hr posttransfection. (B) Expression of telomerase core components. (Top panel) hTR northern blot of total RNA prepared from cell extracts. Actin mRNA served as a loading control. (Bottom panel) Immunoblot of protein extracts probed for hTERT with an hTERT antibody (Rockland). Tubulin served as a loading control. The numbers below the blots indicate the relative amounts of hTR or hTERT normalized to the loading control in each lane and to the WT lane. (C) Telomerase activity in cell extracts as measured by direct elongation of a 5′-biotinylated (TTAGGG)3 primer in the presence of dATP, dTTP, and α-32P-radiolabeled dGTP. Products were purified using magnetic streptavidin beads and resolved on a sequencing gel. RC denotes a recovery control added before DNA purification (5′-biotinylated and 3′-radiolabeled 10-mer oligonucleotide). Relative telomerase activity was obtained by normalizing the 32P incorporation to the RC in each lane and using the standard curve shown in (D). The bottom numbers are the average of activities obtained for all dilutions. Lane 1 corresponds to the primer radiolabeled at the 3′ end with 32P-α-ddATP and terminal transferase, as a +1 size marker. (D) Standard curve of telomerase activity for the WT control, showing initial velocity conditions. Data points in the graph are from (C), lanes 2–7. Molecular Cell 2007 27, 882-889DOI: (10.1016/j.molcel.2007.07.020) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Assembly of Active Telomerase Occurs in Cells, but Not in Cell Extracts HEK293T cells were transiently transfected with the indicated hTR- and/or hTERT-expressing plasmids. Extracts were prepared 48 hr posttransfection. Mix denotes the use of a mixture of cells obtained from cells transfected separately with hTR or with hTERT. (A) Expression of telomerase core components. (Top panel) hTR northern blot of total RNA prepared from cell extracts or mixed extracts. Actin mRNA served as a loading control. (Bottom panel) Immunoblot of protein extracts probed for hTERT with the purified R484 hTERT antibody. Tubulin served as a loading control. The numbers below the blots indicate the relative amounts of hTR or hTERT normalized to the loading control in each lane and to the WT lane. Note that, as a consequence of the mixing, the levels of hTR and hTERT in the mixed extract are half that obtained in extracts from cells expressing only hTR or only hTERT. (B) Telomerase activity in cell extracts as measured by a real-time PCR-based telomerase assay (RQ-TRAP). Bars represent the means ± SD of at least three measurements. Molecular Cell 2007 27, 882-889DOI: (10.1016/j.molcel.2007.07.020) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 A Mutation in the CAB Box Prevents Efficient Telomere Elongation without Affecting Accumulation of Active Telomerase in HeLa Cells HeLa cells were transduced with retroviral vectors expressing hTR or/and Myc-hTERT and selected. Cell populations were used. (A) Expression of telomerase core components. (Top panel) hTR northern blot of total RNA. Actin mRNA served as a loading control. (Bottom panel) Immunoblot of protein extracts probed for hTERT with an hTERT antibody (Rockland). Tubulin served as a loading control. The numbers below the blots indicate the relative amounts of hTR or hTERT normalized to the loading control in each lane and to the WT lane. (B) Telomerase activity in cell extracts as measured by a real-time PCR-based telomerase assay (RQ-TRAP). Bars represent the means ± SD of at least three measurements. (C and D) Telomere-extension assay. DNA was harvested at the indicated population doublings (PD), digested with HinfI and RsaI, and resolved by pulse-field gel electrophoresis. After in situ denaturation, telomeric DNA was detected by in-gel hybridization with a random radiolabeled telomeric probe. (C) Genomic blot of telomeric restriction fragments (TRFs) of cells transduced with the indicated constructs. Steady-state extension rates are indicated below in base pairs per PD (bp/PD) for this gel and were deduced by linear regression of experimental data between PD = 10 and 30. (D) The graph shows median TRF length at the indicated cell population doubling. Molecular Cell 2007 27, 882-889DOI: (10.1016/j.molcel.2007.07.020) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Mutations in the CAB Box Prevent Efficient Telomere Elongation without Affecting Accumulation of Active Telomerase in HT1080 Cells HT1080 cells were transduced with retroviral vectors expressing only WT hTR or the CAB box mutants and were selected. Cell populations were used. (A) Telomerase activity in cell extracts as measured by a real-time PCR-based telomerase assay (RQ-TRAP). Bars represent the means ± SD of at least three measurements. (B and C) Telomere-extension assay. (B) Genomic blot of telomeric restriction fragments (TRFs) of cells transduced with the indicated constructs. Blot was performed as indicated in the legend of Figure 3. Steady-state extension rates are indicated below in base pairs per PD (bp/PD) for this gel and were deduced by linear regression of experimental data between PD = 5 and 37. (C) The graph shows median TRF length at the indicated cell population doubling. Molecular Cell 2007 27, 882-889DOI: (10.1016/j.molcel.2007.07.020) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Reduced Accumulation of hTERT at Telomeres When Assembled with CAB Box Mutant hTR (A) Telomeric chromatin immunoprecipitations (ChIP) were performed using HEK293T cells transfected with the indicated constructs. The antibodies used for IP are indicated on the left. Duplicate dot blots were probed for telomeric or Alu repeats. (B) Quantification of the data in (A). Relative telomerase association with telomeres was calculated by normalizing the percentage of telomeric DNA recovered in each ChIP normalized to that recovered in cells transfected with hTERT and WT hTR. Bars represent the means ± SEM of three independent experiments (n = 3), except for cells transfected only with WT hTR (n = 2). One-tailed paired t tests indicate a statistically significant reduction of telomerase association with telomeres for the m1 mutant as compared to WT (p < 0.05), but not for m2 mutant as compared to WT (p = 0.23). Molecular Cell 2007 27, 882-889DOI: (10.1016/j.molcel.2007.07.020) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Reduced Accumulation of CAB Box Mutant hTR at Telomeres (A) hTR FISH (red) and TRF2 immunostaining (green) were performed on the same stable HeLa cell populations as used in Figure 3 for the telomere length analysis. Telomeres (detected via TRF2) where hTR is colocalized are indicated with white arrowheads in the merge panels. “Merge 2” panel shows an additional cell. (B) Frequency of localization of hTR alleles to telomeres. The number of cells in which the indicated number of colocalizations occurred is expressed as a percentage of the total cells counted in two experiments (n) for the corresponding hTR allele in panel (A). Molecular Cell 2007 27, 882-889DOI: (10.1016/j.molcel.2007.07.020) Copyright © 2007 Elsevier Inc. Terms and Conditions