Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions

Abstract

Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.

FIG. 1

Secondary structure of hTR with its telomeric template sequence (nucleotides 46 to 53) and its 5′ and 3′ ends (adapted from the work of Chen et al. [15]). Four universally conserved structural elements among vertebrate telomerase RNAs, including the pseudoknot, the CR4-CR5 region, the H/ACA box, and the CR7 region, are boxed in gray. The P1, P2a.1, P2a, P2b, and P3 helices are indicated. Arrows indicate the nucleotide positions of the 5′ and 3′ ends of the different hTR truncations used in this study.

FIG. 2

Sequences or structures located between nucleotides 276 and 424 of hTR are necessary for telomerase activity reconstitution in vitro. (A) Northern blot of total RNA harvested from RRLs in which hTERT was synthesized in the absence of hTR (lane 1) or the presence of wild-type hTR (lane 2), hTR(ACA-TGT) (lane 3), or 3′-truncated hTRs (lanes 4 to 9). The Northern blot was probed for hTR-specific sequences. M, DNA markers (sizes, in base pairs, are on the left). RNAs were separated by electrophoresis on a 4% acrylamide–7 M urea gel. (B and C) Equal volumes of RRL reaction products generated in the absence (−) or presence of the different hTR variants were subjected to immunoprecipitation (IP) with an antibody to the T7 tag. The washed beads were analyzed for telomerase activity (B) and hTERT-hTR coimmunoprecipitation (C). For panel B, telomerase activity was analyzed by the TRAP assay, and 100 ng of partially purified 293 cell extracts was used as a positive control (lane 19). IC, internal PCR control; WT, wild-type. For panel C, hTERT-hTR coimmunoprecipitation was analyzed by Northern blotting using an hTR-specific probe. The arrowheads indicate the positions at which hTR1-451 and hTR1-159 migrate.

FIG. 3

Role of the P3 helix of the hTR pseudoknot in telomerase function and two independent hTERT binding sites within hTR. (A) Northern blot of total RNA harvested from RRLs in which hTERT was synthesized in the absence of hTR (lane 1) or presence of wild-type hTR (lane 2) and hTR variants. The Northern blot was probed for hTR-specific sequences. M, DNA markers (sizes, in base pairs, are on the left). RNAs were separated by electrophoresis on a 6% acrylamide–7 M urea gel. (B and C) Equal volumes of RRL reaction products generated in the absence (lanes 11 and 22) or presence of the different hTR variants were subjected to immunoprecipitation (IP) with an antibody to the T7 tag. The washed beads were analyzed for telomerase activity (B) and hTERT-hTR coimmunoprecipitation (C). For panel B, telomerase activity was analyzed by the TRAP assay, and 100 ng of partially purified 293 cell extracts were used as a positive control (lane 21). IC, internal PCR control; WT, wild-type. For panel C, hTERT-hTR coimmunoprecipitation was analyzed by Northern blotting using an hTR-specific probe. The arrowheads indicate the positions at which full-length hTR (FLhTR), hTR164-330, and hTR33-147 migrate.

FIG. 4

Summary of hTERT truncations analyzed and their telomerase activity and extent of binding to hTR. Schematic representation of the human telomerase RT with the seven conserved RT-like motifs (1, 2, A, B′, C, D, and E) as well as the T motif (44). Amino- and carboxy-terminal deletions in hTERT (including amino acid positions) are indicated along with the relative telomerase activities and hTR binding abilities these proteins demonstrated after their immunoprecipitation from RRLs in which they were expressed in the presence of recombinant hTR. All these proteins were engineered to express an N-terminal epitope tag (T7 tag).

FIG. 5

The polymerization and RNA binding functions of hTERT are independent in vitro. (A) Equal volumes of lysate in which the different hTERT proteins (indicated above each lane) were synthesized in the presence of recombinant hTR and [³⁵S]methionine were analyzed for protein expression by 7.5% SDS-PAGE. The gel was dried and exposed to a phosphorimager screen. FLhTERT, full-length hTERT; CT, C-terminal; NT, N-terminal; MOT, motif; M, protein markers (masses, in kilodaltons, are on the right). Equal volumes of the different RRL reaction products were subjected to immunopurification using the T7 tag antibody. The washed beads were analyzed for protein levels using 7.5% SDS-PAGE (B), telomerase activity (C), and hTERT-hTR coimmunoprecipitation (D). (C) Telomerase activity was analyzed by the TRAP assay, and 100 ng of partially purified 293 cell extracts was used as a positive control (lane 23). IC, internal PCR control. (D) hTERT-hTR coimmunoprecipitation was analyzed by Northern blotting using an hTR-specific probe. FLhTR, full-length hTR.