Comprehensive structure-function analysis of the core domain of human telomerase RNA - PubMed (original) (raw)

Comparative Study

Comprehensive structure-function analysis of the core domain of human telomerase RNA

Hinh Ly et al. Mol Cell Biol. 2003 Oct.

Abstract

Telomerase is a cellular reverse transcriptase that uses part of its integral RNA (called TER) as the template to synthesize telomeric DNA repeats. Vertebrate TERs are thought to share a conserved, highly structured core domain that includes the templating sequence and a pseudoknot, but not all features of the predicted core structure have been verified directly or shown to affect telomerase enzymatic activity. Here, we report a systematic mutational analysis of the core domain (residues 1 to 210) of human telomerase RNA (hTER). Our data confirm that optimal hTER activity requires the integrity of four short helices (P2a.1, P2a, P2b, and P3) which create the proposed pseudoknot and that features of both the primary sequence and secondary structure in P2b and P3 contribute to optimal function. At least part of the long-range P1 pairing is also required, despite the lack of a known P1 counterpart in rodent TERs. Among the predicted single-stranded regions, we found that J2b/3, portions of J2a/3, and residues in and around the template make sequence-specific contributions to telomerase function. Additionally, we provide evidence that naturally occurring hTER sequence polymorphisms found in some patients with aplastic anemia can inhibit telomerase activity by disrupting critical structures within the hTER core domain.

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Figures

FIG. 1.

FIG. 1.

(A) Schematic view of the consensus secondary structure of the core 210-nucleotide domain of hTER RNA as proposed by Chen et al. (9). The putative P and J regions are indicated by brackets, and the templating sequence is shown as a solid rectangle. Residues are numbered with respect to the transcriptional start site of the hTER gene. (B and C) Summary results of semiquantitative (TRAP) analyses of telomerase enzymatic activity in reconstituted VA13 cells for various mutations (indicated in boldface) targeting individual paired regions P1, P2, P2a, P2b, P2a.1 and P2a.1ext (B) and sequential deletions of the 5′ terminus (C). The telomerase enzymatic activity of each mutant is expressed in comparison to that of the wild type (+++, 20 to 100%; ++, 2 to 20%; +, 1 to 2%; −, undetectable), based on at least two or three independent determinations that each involved at least three sequential dilutions of extract from the transfected cells.

FIG. 2.

FIG. 2.

Representative TRAP gels illustrating the relative telomerase activities obtained from representative substitution and compensatory mutations in the hTER core domain. The triangles above the lanes indicate serial fivefold dilutions of the cell lysates, with the first lanes of each sample showing telomerase enzymatic activities of approximately equal concentrations of extract made from 2 × 104 cells. WT, wild-type; IC, internal controls for normalization of PCRs. (A) Lanes 5 to 22, aplastic-anemia (AP)-associated polymorphisms in the P3 and P2a.1 stems. (B) Lanes 11 to 18, 5′ deletion mapping of hTER RNA; lanes 8, 9, and 10, control 293T cell lysate, standard telomeric repeats amplified from control TSR8 DNA templates, and heat-inactivated wild-type cell lysate, respectively. All lanes are from the same gel and autoradiogram. (C) Northern blotting analysis of informative hTERs expressed in transfected VA13 cell extracts. The level of hTER RNA was quantified by PhosphorImager image analysis software and is expressed as a ratio of hTER RNA to cellular β-actin mRNA. Lane 9, negative control cell lysate transfected only with hTERT expression construct.

FIG. 3.

FIG. 3.

Summary of semiquantitative TRAP analyses for mutations in the P3 stem. The introduced mutations are highlighted in boldface. The telomerase enzymatic activity of each mutant relative to that of the wild-type hTER is indicated as described in the legend to Fig. 1. (A) Three- and 6-nucleotide substitutions near the 5′ and 3′ termini of the P3 stem. (B) Four-nucleotide substitutions in the center of the P3 stem. (C and D) Dinucleotide substitutions throughout the P3 stem.

FIG. 4.

FIG. 4.

Summary results of semiquantitative (TRAP) analysis of telomerase enzymatic activities in reconstituted VA13 cells for various substitution mutations within the indicated J regions. The telomerase enzymatic activity of each mutant is expressed in comparison to that of the wild type (+++, 20 to 100%; ++, 2 to 20%; +, 1 to 2%; −, undetectable), based on at least two or three independent determinations that each involved at least three sequential dilutions of extract from the transfected cells.

FIG. 5.

FIG. 5.

(A) Representative TRAP gel of three aplastic-anemia-associated hTER polymorphisms. WT, wild-type; IC, internal controls for normalization of PCRs. (B) Northern blot analysis of the representative aplastic anemia-associated hTER RNAs. The level of hTER RNA was quantified by PhosphorImager image analysis software and is expressed as a ratio of hTER RNA to cellular β-actin mRNA. (C) Telomerase enzymatic activities of the aplastic-anemia-associated hTER polymorphisms, which are highlighted in boldface. The activity of each mutant was assayed by TRAP and is expressed as described in the legend to Fig. 1. Deletions are indicated by dashed lines.

FIG. 6.

FIG. 6.

Coimmunoprecipitation of wild-type (WT) and mutant hTERs with epitope-tagged hTERT protein expressed in a rabbit reticulocyte lysate. RNA-protein complexes were precipitated with a FLAG-specific antibody and then analyzed by polyacrylamide gel electrophoresis and probed for hTER sequences. The negative control (−) was a lysate that did not contain the hTERT expression vector.

FIG. 7.

FIG. 7.

Functional topology of the hTER core domain. In this schematic view of the 210-base RNA core, the indicated regions of primary sequence (red) and base-pairing interactions (light blue) were shown in the present study to contribute to optimal telomerase function in cells. The core conformation depicted is identical to that proposed by Chen et al. (9), except that the P2a.1 helix has been extended proximally by 3 bp, as indicated by the results of the present study (Fig. 1C) and others (1). Mutagenesis of the hTER template and its flanking sequences has been reported elsewhere (10, 17). The resolution of the present analysis was limited by the sizes of the mutations used, which generally altered 2 to 9 bases simultaneously; hence, not every indicated base or base pair may be individually required for optimal function. As few as 4 bp in the long-range P1 helix can support wild-type telomerase activity (Fig. 1C and 2B).

References

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