Human Telomerase Activation Requires Two Independent Interactions between Telomerase RNA and Telomerase Reverse Transcriptase  James R. Mitchell, Kathleen Collins  Molecular Cell  Volume 6, Issue 2, Pages 361-371 (August 2000) DOI: 10.1016/S1097-2765(00)00036-8

Figure 1 Secondary Structure of the hTR H/ACA Domain (nt 211–451) Residues mutagenized in this study are highlighted. Δ1-Δ4 indicate deletions, with the sequence above the line replaced by the stable tetraloop sequence GAAA. Conserved elements of the H/ACA motif include the 5′ and 3′ potential pseudouridylation pockets, 5′ and 3′ terminal stems, and H and ACA boxes. The position of the 20 nt inserted sequence tag is indicated by a filled carat between nt 354 and nt 355. Filled ovals indicate conserved base pairs supported by phylogenetic comparison (Chen et al. 2000); hollow ovals indicate putative base pairs. Molecular Cell 2000 6, 361-371DOI: (10.1016/S1097-2765(00)00036-8)

Figure 2 In Vivo Mutational Analysis of the hTR H/ACA Domain (A) Northern blot of total RNA prepared from 293 cells transiently cotransfected with recombinant hTRs and recombinant U64 as a control for transfection efficiency (TC). The blot was probed for the recombinant hTR sequence tag and U64. Endogenous (e)U64 was additionally monitored as an RNA recovery control. Asterisks indicate a putative polyadenylated, 3′ unprocessed precursor form of hTR in each sample. Full-length (FL) hTR transcribed in vitro and in vivo migrates as a doublet. The independently stable hTR H/ACA domain derives from 5′ processing to the boundary of the H/ACA motif (nt 211–451). M, in vitro transcribed recombinant hTR marker. (B) TRAP assay of extracts of 293 cells transiently transfected with the indicated hTR constructs. Each pair of lanes is numbered and represents a single 5-fold dilution series; the single lane 16 reaction contained buffer alone. PCR amplification of the altered-template sequence yields a single product. Molecular Cell 2000 6, 361-371DOI: (10.1016/S1097-2765(00)00036-8)

Figure 3 In Vivo Association of hTR with Dyskerin or TERT Northern blot of HA (upper) and FLAG (lower) immunopurification supernatants (S) and bound fraction pellets (P). Extracts were made from 293 cells transiently cotransfected with HA-dyskerin, FLAG-TERT, and recombinant (r)hTR as indicated. Pellets were loaded at 2× relative concentration of supernatants. The blot was probed for the recombinant hTR sequence tag, and regions of the gel corresponding to full-length (FL) hTR or the hTR H/ACA domain are indicated. Asterisks in lane 13 indicate an unprocessed form of hTR. Molecular Cell 2000 6, 361-371DOI: (10.1016/S1097-2765(00)00036-8)

Figure 4 Independent Association of the hTR H/ACA Domain with TERT in Cells that Lack Endogenous hTR Northern blot of FLAG immunopurification supernatants (S) and pellets (P) from extracts of VA13 cells transiently cotransfected with FLAG-TERT and the indicated recombinant (r)hTR. Pellets were loaded at 2× relative concentration of supernatants. The blot was probed for the recombinant hTR sequence tag and endogenous (e)U64 as a control for immunopurification specificity. FL hTR, full-length hTR; M, in vitro transcribed recombinant hTR marker. Molecular Cell 2000 6, 361-371DOI: (10.1016/S1097-2765(00)00036-8)

Figure 6 Mutational Analysis of the IH1 Element (A) Potential secondary structure of the hTR IH1 element. Filled ovals indicate phylogenetically conserved base pairs; hollow ovals indicate putative base pairs. Asterisks designate residues conserved in all known vertebrate telomerase RNAs, and the previously defined CR4-CR5 region is indicated (Chen et al. 2000). Substituted and deleted residues are indicated and highlighted. (B) TRAP assay of immunopurified pellets from (C). Endogenous (WT) and recombinant (AT) activities were assayed for each immunopurified sample over a series of two or three 5-fold dilutions, respectively. Reactions in lanes 1 and 2 contained no extract. A competitive PCR internal control (IC) was included in the WT reactions. (C) Northern blot of FLAG immunopurification supernatants (S) and pellets (P) from extracts of 293 cells transiently cotransfected with FLAG-TERT and the indicated hTR construct. Pellets were loaded at 2× relative concentration of supernatants. The blot was probed for the recombinant hTR sequence tag and endogenous (e)U64 as a control for immunopurification specificity. Regions of the gel corresponding to full-length (FL) hTR and the hTR H/ACA domain are shown. M, in vitro transcribed recombinant hTR marker. Molecular Cell 2000 6, 361-371DOI: (10.1016/S1097-2765(00)00036-8)

Figure 5 IH1 Rescues Telomerase Activity and TERT Association of the hTR-U64 Chimera (A) TRAP assay of immunopurified pellets from 293 or VA13 cells transiently cotransfected with FLAG-TERT and recombinant full-length (FL) hTR or the hTR chimera as indicated. All recombinant RNAs contained the altered template. PCR amplification of telomerase extension products templated by the altered template (AT) yields a single product while amplification of wild-type telomeric repeats templated by the endogenous enzyme (WT) yields a ladder of bands. A competitive PCR internal control (IC) was included in the WT reactions. Triangles represent extract dilutions, in two or three 5-fold steps as indicated. (B) Northern blot of FLAG immunopurification supernatants (S) and pellets (P) from extracts of 293 or VA13 cells transiently cotransfected with FLAG-TERT and the indicated recombinant RNA. Pellets were loaded at 2× relative concentration of supernatants. The blot was probed for the recombinant hTR sequence tag and U64 nt 66–134. Full-length (FL) RNA products are indicated. Asterisks denote the unprocessed recombinant RNA in each lane. M, in vitro transcribed recombinant RNA markers as indicated. Molecular Cell 2000 6, 361-371DOI: (10.1016/S1097-2765(00)00036-8)

Figure 7 In Vitro Requirements for Telomerase Activity (A) TRAP assay of telomerase activity reconstituted in vitro in rabbit reticulocyte lysate (RRL) with HA-TERT and 500 nM final assembly reaction concentration of the indicated in vitro transcribed RNA. Each reconstitution reaction was assayed in a 5-fold dilution series. Mock reactions (lanes 1 and 2) contained HA-TERT but no RNA. (B) TRAP assay of telomerase reconstituted in vitro with HA-TERT in RRL and the indicated hTR fragment. Each triangle represents an RNA titration from 1 nM to 1 μM in the assembly reaction as indicated in the box. A competitive PCR internal control (IC) was added to ensure that the observed telomerase activity was not saturated under the PCR conditions used. (C) Role of order of addition of hTR fragments on transactivation in vitro. The indicated hTR fragment was assembled with HA-TERT and then immunopurified on HA antibody resin. Arrows indicate washing steps to remove unbound RNA. In lanes 13–15 and 16–18, a second hTR fragment was assembled and washed as indicated. In lanes 19–21, the two indicated HA-TERT/RNA resins were washed prior to mixing. All washed samples were assayed in a series of three 5-fold dilutions. A competitive PCR internal control (IC) was added to ensure that the observed telomerase activity was not saturated under the PCR conditions used. Molecular Cell 2000 6, 361-371DOI: (10.1016/S1097-2765(00)00036-8)