Telomerase RNA function in recombinant Tetrahymena telomerase - PubMed (original) (raw)
Telomerase RNA function in recombinant Tetrahymena telomerase
J D Licht et al. Genes Dev. 1999.
Abstract
Telomerase is a ribonucleoprotein reverse transcriptase specialized for use of a sequence within its integral RNA component as the template for DNA synthesis. Telomerase adds telomeric simple sequence repeats to single-stranded primers in vitro or chromosome ends in vivo. We have investigated the sequences and structures of recombinant Tetrahymena thermophila telomerase RNA necessary for physical association and activity with the catalytic protein subunit expressed in rabbit reticulocyte lysate. In contrast with previous results using another reconstitution method, we find that phylogenetically conserved primary sequences and a phylogenetically nonconserved secondary structure are essential for telomerase RNA function. Telomerase RNA binding to the catalytic protein subunit requires sequences 5' of the template and is highly sequence specific. Other telomerase RNA sequences are required for enzyme activity and proper template use but not for protein interaction affinity. In addition, we demonstrate that the production of active recombinant telomerase requires a factor in rabbit reticulocyte lysate that promotes ribonucleoprotein assembly. These studies demonstrate multiple functions for the telomerase RNA and indicate that recombinant telomerase activity requires more than the catalytic protein and RNA components of the enzyme that have been identified to date.
Figures
Figure 1
Telomerase RNA in T. thermophila. The sequence and structure of the T. thermophila telomerase RNA are shown as described previously (Greider and Blackburn 1989; ten Dam et al. 1991; Romero and Blackburn 1991; Zaug and Cech 1995). The representation here emphasizes the secondary structure elements and the primary sequences examined in this study. Stem I is in light blue, stem–loop II is in purple, the stem of stem–loop III as initially described is in green (IIIa), with pseudoknot formation producing a second stem in brown (IIIb), and stem-loop IV is in dark blue. The template region is in bold, and phylogenetically conserved sequences in the single-stranded, template-adjacent regions are in red. RNA 5′ and 3′ ends are indicated.
Figure 2
RNA sequence specificity of telomerase RNP reconstitution in lysate. Five microliters of lysate-expressed TERT was supplemented with 500 ng of the indicated competitor RNA and then with 10 ng of telomerase RNA. Samples were incubated at 30°C for 30 min then assayed for telomerase activity with the primer (TG)8TTG. (Lane 1) No competitor; (lane 2) a telomerase RNA variant missing stem–loop IV, which binds TERT but is less active than the wild-type RNA (see Results); (lanes 3,4) tRNA; (lanes 5,6) 5S RNA; (lanes 7,8) total yeast RNA; (lanes 9,10) a 24-nucleotide RNA oligonucleotide representing telomerase RNA residues 36–59 which have no activity (Collins and Gandhi 1998). In some experiments, total yeast RNA stimulated telomerase activity to the same level as tRNA (not shown). Products corresponding to primer +1 and +6 nucleotides are indicated.
Figure 3
Reconstitution by coexpression of telomerase RNA and TERT. Coexpression reactions contained TERT and the indicated telomerase RNA variant. (A) An equal volume of each reaction was assayed for telomerase activity with the primer (TG)8TTG. Products corresponding to primer +1 and +6 nucleotides are indicated. (B) An equal volume of each reaction was analyzed for telomerase RNA by Northern blot hybridization. The asterisk indicates the ∼160-bp DNA fragment encoding the stem–loop IV deleted RNA, which does not encode a full-length telomerase RNA sequence (see Materials and Methods). (C) An equal volume of each lysate coexpression reaction was analyzed for TERT by SDS-PAGE and autoradiography.
Figure 4
Reconstitution by addition of purified RNA to lysate-expressed TERT. All reactions contained the same TERT expression lysate and the indicated telomerase RNA variant. One hundred nanograms of purified RNA was added to 3 μl of TERT expression lysate, incubated for 30 min at 30°C, then assayed for telomerase activity with the primer (TG)8TTG. RNAs are in ∼40-fold molar excess of TERT. Products corresponding to primer +0, +1, and +6 nucleotides are indicated.
Figure 5
Telomerase RNA binding to TERT. Lysate-expressed, epitope-tagged TERT [(+) TERT lanes] or an equal volume of mock lysate synthesis reaction lacking the plasmid encoding TERT [(−) TERT lanes] was mixed with 100 ng of the indicated telomerase RNA variant. Samples were incubated at 30°C for 30 min after which a fraction of the sample was removed for a telomerase activity assay with the primer (TG)8TTG (A). The remainder of the sample was mixed with HA antibody–protein G–Sepharose. Bound material was washed repeatedly and analyzed by Northern blot hybridization for telomerase RNA (B) and by SDS-PAGE and autoradiography to verify equivalent protein recovery (not shown). Telomerase RNAs with a wild-type 3′ end transcribed from _Fok_I-cut plasmid DNAs with purified T7 RNA polymerase appear as a sharp band and a slightly higher molecular mass RNA smear, likely derived from slippage of the polymerase on the homopolymeric sequence tract at the end of the template. Note that the background of RNA binding to antibody resin in the absence of TERT is minimal (lanes 2,3). Parallel recovery of RNAs was verified by addition of an internal control RNA to samples before RNA extraction (not shown). Immunopurification supernatants were also analyzed by Northern blot hybridization to verify RNA stability during the antibody incubation (not shown).
Figure 6
A role for lysate in recombinant telomerase RNP assembly. (A) Lysate-expressed, epitope-tagged TERT was left alone or immunopurified on HA antibody–protein G–Sepharose either after (lane 1) or before (lanes 3,4) addition of 5 μg total yeast RNA and 100 ng telomerase RNA and incubation at 30°C for 30 min. Telomerase RNA was also added to the original TERT-expression lysate and assembled at the same time as the purified TERT samples (lane 2). Two microliters of fresh lysate was added to the sample in lane 4 before incubation. Samples were assayed for telomerase activity with the primer (TG)8TTG. (B,C) Lysate-expressed, epitope-tagged TERT (lanes 1,2) and a mock synthesis reaction lacking the TERT expression plasmid (lane 3) were immunopurified on HA antibody–protein G–Sepharose. Samples were supplemented with 5 μg of total yeast RNA, 100 ng of telomerase RNA, and where indicated with 2 μl of fresh lysate before assembly at 30°C for 30 min. Half of each sample was analyzed for telomerase activity with the primer (TG)8TTG (B) and half was washed thoroughly and analyzed by Northern blot hybridization for telomerase RNA that remained associated with antibody-bound TERT (C).
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