Biological and biochemical functions of RNA in the tetrahymena telomerase holoenzyme

Abstract

Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.

FIG. 1.

Structure and expression of TER. (A) Selected stages of the telomerase catalytic cycle. Internal template boundaries are influenced by TERT-TER interactions that restrict template motion, illustrated here as small ovals. The default positioning of template may favor placement of TER position C49 in the active site (top). With hybrid formation (middle), the template can be pulled through the active site and used to direct dNTP addition until the 5′ boundary is reached at position C43 (bottom). (B) Schematic of eTER and hpTERs. Predicted eTER secondary structure elements are indicated by roman numerals. (C) Total cellular RNA was analyzed by Northern blot hybridization using oligonucleotides complementary to TER and SRP RNA.

FIG. 2.

Function of hpTER. (A) Genomic DNA was digested with HindIII and analyzed by Southern blotting from the parental strain (lane 1), the hpTER strain coexpressing eTER (lane 2), or the hpTER strain with TER gene disruption (lane 3). The hybridization probe had identical homology with endogenous and disrupted TER loci, which were distinguishable by restriction fragment length as indicated. (B and C) Cell extracts normalized for total protein concentration (inputs) were used to purify hpTER-tagged telomerase RNP (elutions). For TER analysis by Northern blot hybridization in panel B, inputs represent ∼5% of the total amount of cell extract used for binding. Aliquots of the elution mixtures were assayed for activity in panel C with the primer (G₄T₂)₃ in reaction mixtures with 0.6 μM [³²P]dGTP. The first seven dNTP additions are indicated along with a schematic of sequential repeat additions.

FIG. 3.

Expression of hpTER variants. (A) Sequence substitutions are depicted within boxes expanded from the schematic. Nucleotide positions are numbered in the eTER context. (B and C) RNA was prepared from equal numbers of cells (B) or from cell extracts (C) and analyzed by Northern blot hybridization. In panel C, levels of hpTER relative to eTER are indicated after normalization to SRP RNA as a loading control.

FIG. 4.

In vitro reconstitution of TERT and hpTER in RRL. Two microliters of TERT protein expression reaction mixture was combined with 1 pmol of TER prior to RNP assembly for 15 min at 30°C, as done previously (25, 28, 35). Samples were then diluted 10-fold into T2MG buffer to provide half of the final activity assay reaction volume. Assay mixtures contained 5.6 μM dGTP and 400 μM dTTP for optimal activity of the minimal recombinant RNP (16). (A and B) Reconstituted wild-type TER (lanes 1), wild-type hpTER (lanes 2), and hpTER variants were assayed using either 250 nM of the telomeric repeat primer (GT₂G₃)₃ (A) or 250 nM of (TG)₈T₂G₃ (B), a primer optimal for the minimal recombinant RNP. (C) Reconstituted wild-type TER (lanes 1 and 2), wild-type hpTER (lanes 3 and 4), and the hpTER variant UCA38-40AGU (lanes 5 and 6) were assayed using 250 nM (TG)₈T₂G₃ in reaction mixtures without (-) or with (+) 400 μM dATP. The dATP-dependent change in product profile for the TBE variant enzyme occurs due to synthesis past the normal template 5′ boundary to copy TER position U42 (aberrant template position +7).

FIG. 5.

Telomere length maintenance by telomerase holoenzymes reconstituted with hpTER variants. Genomic DNA was digested with HindIII, resolved by denaturing polyacrylamide gel electrophoresis, and assayed by Southern blotting for telomeric restriction fragments containing the rRNA chromosome subtelomeric region. End-labeled DNA marker (M) lengths are indicated at right. (A) Cells were grown for a fixed number of doublings at 30°C. (B) Cells were grown in continuous log-phase culture at 30°C with aliquots harvested for analysis at days 3, 5, and 7. All strains doubled at similar rates.

FIG. 6.

Catalytic activity of telomerase holoenzymes containing hpTER variants. All wild-type (WT) samples refer to hpTER with no additional sequence substitutions. (A) Eluted samples were assayed for activity as indicated with the primer (GT₂G₃)₃, which is elongated by addition of the sequence GTTG to reach the template 5′ end at position +6. The product ladder is annotated to indicate complete repeat additions. Lane 17 is another activity assay using RNP with the TBE variant UCA38-40AGU in dTTP; this relatively long exposure revealed repeat addition processivity. (B) TERT and p65 were detected in purified samples by immunoblotting using rabbit polyclonal antibodies raised and affinity purified against a TERT peptide or full-length p65 (42). Neither protein was recovered by the control affinity purification from a cell extract lacking hpTER (mock). Note that differences in recovery do not reflect relative differences in RNP stability or purification yield because extracts were not normalized for hpTER prior to purification. (C) Successive complete-repeat addition products from parallel assays of wild-type or TRE and pseudoknot substitution hpTER RNPs were quantified for intensity, normalized for specific activity, and plotted to compare repeat addition processivities.

FIG. 7.

Template boundary definition by the TBE. (A) Schematic of products produced in panels B and C by enzymes with TBE substitutions. TER positions 41 to 51 and primer sequence are shown in uppercase letters; nucleotides added during product synthesis are shown in lowercase letters. (B) Eluted samples were assayed for activity as indicated with the primer (GT₂G₃)₃, which was elongated by addition of GTTG to reach the template 5′ boundary at position +6. The product ladder is annotated to indicate complete repeat additions. Synthesis past the normal template 5′ boundary to copy TER position U42, indicated as +7 product, was detected in reactions with dATP. (C) Eluted samples were assayed for activity as indicated with 1 μM primer (G₃T₂G)₃, which can be elongated by dGTP addition at template position +1 or by dATP addition at aberrant template position +7. Either dGTP (dG*) or dATP (dA*) was radiolabeled. The dG* reaction mixtures contained 0.3 μM radiolabeled dGTP and 1 μM unlabeled dGTP, with 400 μM dTTP and dATP if indicated. Reactions in lanes 3 to 5, 9 to 11, and 15 to 17 contained, respectively, 0.3, 0.6, and 1.3 μM radiolabeled dATP mixed with unlabeled dATP to provide a total of 1.3 μM; these reaction mixtures also contained 0.3 μM dGTP and 400 μM dTTP. Reaction mixtures for lanes 6, 12, and 18 contained 1.3 μM radiolabeled dATP as the only nucleotide substrate.

FIG. 8.

A TRE role in use of the template 3′ end. (A) Eluted samples were assayed for activity as indicated with the primer (GT₂G₃)₃. The product ladder is annotated to indicate sequential repeat additions to template position +6 or products from synthesis to template position +2. (B) The intensity of each product within the first five repeats in odd lanes of panel A was adjusted for specific activity and then normalized as the percentage of total product within the repeat. A schematic of the template position numbers is shown.

FIG. 9.

A kinetic defect imposed by substitution of the TRE. (A and B) RNPs were immobilized on IgG-agarose using TAP-tagged coat protein. Bead-bound enzymes were assayed with the primer (GT₂G₃)₃, 400 μM dTTP, and 0.6 μM radiolabeled and 1 μM unlabeled dGTP for 2.5 min at 30°C. Reactions were stopped (lanes 1 and 11), and bound products were retained after washes to remove unbound primer and product (lanes 2 and 12). After an additional 15-min incubation at 30°C, beads were again washed to detect stably bound products (lanes 3 and 13). Extension of bound products was then assayed by addition of unlabeled 1 μM dGTP and 400 μM dTTP (lanes 4 and 14, time zero) in the absence or presence of 1 μM or 5 μM of the primer (G₃T₂G)₃ as depicted by the black triangle. Chase reactions were halted after 15 min at 30°C followed by separation of bead-associated (lanes 5 to 7 and 15 to 17) or released (lanes 8 to 10 and 18 to 20) products. (C) IgG-agarose was preincubated without (-) or with (+) cell extract and washed, followed by addition of telomerase products purified after a 2.5-min activity assay. Samples were incubated for 20 min at 30°C followed by separation of products that became bead associated (B, odd lanes) or remained in solution (S, even lanes).