Identification of Functional Domains and Dominant Negative Mutations in Vertebrate Telomerase RNA Using an in Vivo Reconstitution System*

Luis Martín-RiveraDagger and María A. Blasco§

From the Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Campus Cantoblanco, E-28049 Madrid, Spain

Received for publication, September 14, 2000, and in revised form, October 24, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The telomerase holoenzyme consists of two essential components, a reverse transcriptase, TERT (telomerase reverse transcriptase), and an RNA molecule, TR (telomerase RNA, also known as TERC), that contains the template for the synthesis of new telomeric repeats. Telomerase RNA has been isolated from 32 different vertebrates, and a common secondary structure has been proposed (Chen, J.-L., Blasco, M. A., and Greider, C. W. (2000) Cell 100, 503-514). We have generated 25 mutants in the four conserved structural domains of the mouse telomerase RNA molecule, mTR, and assayed their ability to reconstitute telomerase activity in mTR-/- cells in vivo. We found that the pseudoknot and the CR4/CR5 domains are required for telomerase activity but are not essential for mTR stability in the cell, whereas mutations in the BoxH/ACA and the CR7 domains affect mTR accumulation in the cell. We have also identified mTR mutants that are able to inhibit wild type telomerase in vivo.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Telomerase is a reverse transcriptase that synthesizes telomeric repeats onto chromosome ends (2-5). The catalytic protein subunit, TERT,1 shares sequence features with reverse transcriptases (6-12). TERT is tightly associated with an RNA molecule, TR, that provides the template for the synthesis of new telomeric repeats (5, 13). Several associated proteins interact with the TR and TERT components of the telomerase holoenzyme (14-17). The telomerase RNA molecule has been isolated from ciliates, yeasts, and mammals and shown to be essential for telomerase activity (13, 18-20). The telomerase RNAs from ciliates range in size from 148 to 209 nucleotides and share a conserved secondary structure (21-23). In vertebrates, telomerase RNAs are longer, from 382 to 559 nucleotides (1, 18, 19, 24), and in yeast, they are 1200-1300 nucleotides in length (20, 25). Several elements of secondary structure have been described both in mammals and in yeasts (26, 27). In particular, a small nucleolar RNA BoxH/ACA structure has been identified at the 3' end of mTR and hTR and found to be important for TR stability and processing in vivo (26). Several structure-function analyses of the full-length human telomerase RNA, hTR, have been carried out using various in vitro reconstitution assays (28-30). Importantly, a secondary structure for vertebrate TRs has been recently determined by phylogenetic comparative analysis (1). In this study it is proposed that all vertebrate telomerase RNAs share four highly conserved structural domains as follows: a pseudoknot domain, a CR4-CR5 domain, a BoxH/ACA domain, and a CR7 domain (1). Here we address the role of the four conserved domains in vertebrate telomerase RNA by assaying the ability of mTR mutations to reconstitute telomerase activity in mTR-/- cells in vivo (31). In contrast to previous studies in mammals, mutant mTRs were expressed under the endogenous mouse telomerase gene promoter in an in vivo reconstitution system. In our in vivo reconstitution analysis, we find regions of mTR that are important for telomerase activity, RNA accumulation, and processing in vivo, and we map these regions onto the recently proposed secondary structure of vertebrate telomerase RNA (1). Furthermore, we identified mTR unpaired regions in the template, the pseudoknot and the CR4-CR5 domains, which are potential targets for telomerase inhibition.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- All mutants were generated using as template a plasmid with 1.9 kb of mouse genomic DNA containing the wild type mTR gene under its own promoter sequences. This plasmid was constructed by digesting a 5-kb EcoRI genomic fragment containing the mTR locus (18) with XbaI and EcoRV and then subcloning the resulting 1.9-kb fragment in a pBluescript SK-.

Mutagenesis-- To generate deletion and substitution mutants, we used the Sculptor in vitro mutagenesis system from Amersham Pharmacia Biotech (32). All mutants were sequenced using an ABI 377 DNA Sequencer and the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer Life Sciences). The following oligonucleotides were used for mutagenesis: Delta 3-10, 5'GGGTATTTAAGGTCGAGGGCGGCTAGGCCTCGGCACGATTTTCATTAGCTGTGGGTTCTGG3'; Delta 23-102, 5'CACCTAACCCTGATTTTCATTAGCGGGCCACCGCGCGTTCCCG3'; Delta 106-185, 5'GAAAGTCCAGACCTGCAGCGGCGCCCAGTCCCGTACCCGCCTACAGGCCG3'; Delta 199-294, 5'GCCGGGCCGCCCAGTCCCGTCCAGGCCGGGCGAGCGCCGCG3'; Delta 328-389, 5'GCGCCGCGAGGACAGGAATGTCACAACCCCCATTCCCGCTG3'; Delta 56-73, 5'GGTCTTTTGTTCTCCTCCGCCCGCTGCAGCGGGCCAGGAAAGTCCAG3'; Delta 120-143, 5'CCAGACCTGCAGCGGGCCACCGCGCGTTCCGCAGGAGCTCCAGGTTCG3'; Delta 154-167, 5'CCTCAAAAACAAACGTCAGCGCAGGAGCTAGCTCCGCGGCGCCGGGCCGCCCAGTCCCG3'; Delta 220-241, 5'CCGTACCCGCCTACAGGCCGCGGCCGGCCCTGCCGCCGCGAAGAGCTCGCCTCTGTCAGC3'; Delta 254-266, 5'GGGTCTTAGGACTCCGCTGCCGCCGCGAGTCAGCCGCGGGGCGCCGGGGGCTG3'; Delta 357-364, 5'GGTCCCCGTGTTCGGTGTCTTACCGGGAAGTGCACCCGGAACTCGGTTC3'; Delta 341-345, 5'GGACAGGAATGGAACTGGTCCCCGGGTGTCTTACCTGAGCTGTGG3'; Delta 31-3, 5'CCCTGATTTTCATTAGCTGTGGGTCTTTTGTTCTCCGCCCGCTGTTTTTC3'; Delta 90-94, 5'GCTGACTTTCCAGCGGGCCAGGAAAGACCTGCAGCGGGCCACCGCGTTCCCGAGCCTC3'; substitution 57-61 U right-arrow A, 5'GGTCTTTTGTTCTCCGCCCGCTGAAAAACTCGCTGACTTCCAGCGGGCCAGGAAAGTCC3'; substitution 129-133 A right-arrow U, 5'CACCGCGCGTTCCCGAGCCTCTTTTTCAAACGCAGCGCAGGAGCTCC3'; substitution 65-70 GCUGAC right-arrow GUCAGC, 5'GGTCTTTTGTTCTCCGCCCCTGTTTTTCTCGTCAGCTTCCAGCGGGCCAGGAAAGTCCAG3'; substitution 139-144 GUCAGC right-arrow GCUGAC, 5'-GTTCCCGAGCCTCAAAAACAAACCTGACGCAGGAGCTCCAGGTTC3'; substitution 71-74 UUCC right-arrow GGAA, 5'GGTCTTTTGTTCTCCGCCCGCTGTTTTTCTCGCTGACGGAAAGCGGGCCAGGAAAGTCCAG3'; substitution 71-74 + 84-87 UUCC right-arrow GGAA + GGAA right-arrow UUCC, 5'GTTTTTCTCGCTGACGGAAAGCGGGCCATTCCAGTCCAGACCTG3'; box H mutant A319U/ A321U/A324U, 5'CAGGCCGGGCGAGCGCCGCAAGGTCTGGTATGGAACTGGTCCCCGTG3'; box ACA mutant A392U/A394U, 5'GCACCCGGAACTCGGTTCTCTCTACCCCCATTCCCGCTGGGGAAATGCC3'.

To generate the rest of mTR mutants, we used QuikChange, a PCR-based mutagenesis kit provided by Stratagene. The oligonucleotides used to generate each mutant are as follows: Delta 172-314, forward, 5'CCAGGTTCGCCGGGAGCTCCGAGGACAGGAATGGAACTGG3'; Delta 172-314, reverse, 5'CCAGTTCCATTCCTGTCCTCGGAGCTCCCGGCGAACCTGG3'; substitution 65-72 GCUGACUU right-arrow AAGUCAGC, forward, 5'GGTCTTTTGTTCTCCGCCCGCTGTTTTTCTCCGACTGAACCAGCGGGCCAGGAAAGTCCAG3'; substitution 65-72 GCUGACUU right-arrow AAGUCAGC, reverse, 5'CTGGACTTTCCTGGCCCGCAGGTTCAGTCGGAGAAAAACAGCGGGCGGAGAACAAAAGACC3'; substitution 136-144 AACGUCAGC right-arrow GCUGACGUU, forward, 5'CGTTCCCGAGCCTCAAAAACATTCCAGTCGGCAGGAGCTCCAGGTTCG3'; substitution 136-144 AACGUCAGC right-arrow GCUGACGUU, reverse, 5'CGAACCTGGAGCTCCTGCCGACTGGAATGTTTTTGAGGCTCGGGAACG3'.

Dominant Negative Clones-- A NotI-XbaI fragment containing a neo cassette was subcloned in the plasmids containing the selected mTR mutants (see Fig. 5) and in the empty vector (pBluescript SK-, see above). Immortal fibroblasts, derived from wild type embryos (31), were transfected with 20 µg of each of the constructs by conventional calcium phosphate transfection. After 2 days, 1/100, 1/50, 1/20, and 1/10 dilutions were plated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and G418 (200 µg/ml). After 2 weeks of selection several stable clones were picked from the 1/100 and 1/50 dilutions and expanded in selective medium. S-100 extracts, TRAP assays, and telomerase activity quantifications were performed as described below.

Transfection of mTR-/- Cells for Reconstitution Assays-- KO3 p23 cells were transfected with 20 µg of each plasmid using a conventional calcium phosphate protocol (31). As control for transfection efficiency, cells were co-transfected with 1 µg of a LacZ expression vector (J7Laz). The transfection efficiency of the lacZ gene was measured by absorbance at 420 nm after addition of O-nitrophenyl beta -D-galactopyranoside (Sigma). Cells were divided 48 h post-transfection and further cultured overnight. One dish was used for S-100 extracts and the other for RNA preparation, as described below.

Northern Blot-- Forty eight hours after transfection, total RNA was prepared as described (33). In the case of the stable transfectants total RNA was prepared from 10-cm dishes after clone amplification as described above. Typically, 30 µg of total RNA was used for Northern blots, except for the control (wild type cells, 10 µg). RNA was separated by electrophoresis on a 6% acrylamide/bisacrylamide (19:1), 7 M urea, TBE 0.6× gel for 2 h at 5 watts. After electrophoresis, RNA was transferred to HybondTM-N+ membranes (Amersham Pharmacia Biotech). Blots were probed with radiolabeled probes against mTR and 5 S RNA. RNA levels were quantified using IQMac version 1.2, and the mean of at least two different experiments was calculated. In all cases, mTR levels in the cell were corrected by the efficiency of transfection of a LacZ plasmid. Final values of mTR abundance in the cell were corrected by transcript size. Finally, as loading control, gels were also probed with the 5 S RNA.

Preparation of S-100 Extracts-- S-100 extracts from transient or stable transfectants were prepared as described (31). Protein concentration of extracts was determined using the Bio-Rad Protein Assay (Bio-Rad).

TRAP Assay-- Reconstituted telomerase activity was measured 48 h after transfection in a modified version of the TRAP assay (34, 35). In the case of the stable transfectants, telomerase activity was measured from 10-cm dishes after clone amplification. Telomerase activity levels were quantified using IQMac version 1.2 (the whole telomerase ladder was quantified at the two different concentrations and corrected by dilution, background, and internal control signal). For quantification, films with nonsaturated signals were used. The TRAP results shown in Fig. 3 are for illustrative purposes; some of the films shown are overexposed to allow the visualization of TRAP activity by mutants that are severely affected. The transfection efficiency of the mTR mutants was determined by co-transfecting a plasmid containing LacZ (not shown). In all cases an internal control for PCR efficiency was included in the TRAP assay (TRAPeze kit, Oncor).

Western Blot Analysis Using K-370 Telomerase-specific Antibodies-- One hundred µg of total protein from S-100 extracts (see above) were loaded per lane, and Western blots with K-370 antibody were carried out exactly as described in Martín-Rivera et al. (10). For full characterization of K370 antibody specificity see Martín-Rivera et al. (10).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse Telomerase RNA Secondary Structure-- In all vertebrates, four distinct conserved structural domains have been defined for the telomerase RNA molecule as follows: (i) the pseudoknot domain, (ii) the CR4-CR5 domain, (iii) the BoxH/ACA domain, and (iv) the CR7 domain (1). Fig. 1 shows the conserved secondary structure for vertebrate telomerase RNAs mapped onto the mouse telomerase RNA (mTR). Eight regions of base pairing interactions are proposed for mTR (P2-P8), which define the four conserved structural domains. A region of long range base pairing, P1, which is conserved in many vertebrates, is not found in mouse or other rodents (1) (Fig. 1).



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Fig. 1.   Proposed secondary structure of mouse telomerase RNA. Paired regions (P) are numbered from 5' to 3' as P2-P8 (P1 is missing in mTR). The junction regions (J) between two paired regions are termed with reference to the flanking paired regions. The four universal structural domains conserved in all vertebrate telomerases are shaded in gray and labeled. The template region, Box H, and Box ACA motifs are labeled, and the conserved nucleotides are boxed. The mTR secondary structure model is based on the vertebrate TR structure published by Chen et al. (1).

mTR Stability and Processing in Vivo-- All 25 mTR mutations were obtained in the context of a genomic construct containing the mTR gene under endogenous promoter sequences shown to be sufficient to direct mTR gene transcription in vivo (18, 31). The telomerase RNA null cell line KO3 p23, derived from first generation mTR-/- mouse embryonic fibroblasts, was used for in vivo reconstitution of telomerase activity with the mutant mTRs (31). To study the effect of the mutations on mTR stability in the cell, we performed Northern blot analysis of total RNA isolated from KO3 p23 cells 48 h after transfection with wild type or mutant mTR constructs ("Experimental Procedures"). Fig. 2, a---c, shows representative Northern blot images of mTR accumulation in the cell 48 h after transfection. In mTR-/- cells transfected with wild type mTR, a full-length mTR transcript stably accumulated, showing the same mobility as the endogenous mTR (Fig. 2, a-c, compare mTR band in wild type and control cells lanes). As expected, when the mTR-/- cells were transfected with an empty vector, no mTR-specific signal was detected (see empty vector lane in Fig. 2, a and c).



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Fig. 2.   In vivo stability of mutant telomerase RNAs. a-c, Northern blot analysis of mTR expression in wild type (mTR+/+) and telomerase-deficient KO3 p23 (mTR-/-) cells 48 h after transfection with wild type or mutant mTR constructs. Notice that these Northern blots are shown for illustrative purposes. Detailed quantification of RNA levels for all the mutant mTR is shown in Table I. mTR+/+ cells show the characteristic mTR band (397 nucleotides) and the 5 S ribosomal RNA band that was used as internal control (220 nucleotides). mTR-/- cells show different sized mTR-specific signals after transfection with the corresponding wild type or mutant constructs, as well as the 5 S ribosomal RNA band used as loading control. Note the amount of RNA in mTR+/+ control cells was 10 and 30 µg in the rest. Also note that the amount of radioactivity in the 5 S probe was 10% of that used for mTR to allow visualization of the low abundance of mTR transcripts. The asterisk indicates the aberrant mobility of mutant Delta 254-266.

At least 2 to 3 different transfections were carried out for every mTR mutant, and average mutant mTR abundance in the cell was quantified and corrected by transfection efficiency of a LacZ plasmid (see "Experimental Procedures"). Mutant RNA accumulation values relative to that of the transfected wild type mTR are shown in Table I.


                              
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Table I
Summary of stability and activity of the different mTR versions

The Pseudoknot and CR4-CR5 Domains-- mTR deletions Delta 23-102, Delta 31-36, and Delta 90-94 and all 9 mTR substitutions affecting the pseudoknot domain and the pseudoknot interaction accumulated in the cell at the predicted size to similar levels as the transfected wild type mTR (Fig. 2, a---c, and Table I). Mutants Delta 120-143 and Delta 56-73, which also disrupted the pseudoknot interaction (helix P3), accumulated to 45 and 51% of wild type mTR levels (Fig. 2a and Table I). A large mTR deletion (Delta 106-185) disrupting conserved helices P3 and P4 in the pseudoknot and BoxA/ACA domains, respectively, reduced mTR accumulation to 41% of wild type levels (Fig. 2a and Table I). mTR deletion mutant Delta 154-167, which reduces the distance between the pseudoknot and the Box/ACA and CR4-CR5 domains, showed 47% of wild type levels (Fig. 2a and Table I).

Interestingly, deletion of several conserved nucleotides of the J6/5 region in mutant Delta 254-266 in the CR4-CR5 domain resulted in accumulation of a lower mobility form of mTR (see asterisk in Fig. 2a), this mutant showed 85% of wild type mTR levels (Fig. 2a; Table I). The complete sequencing of all mTR constructs rules out a cloning error as responsible for this higher molecular weight mTR (see "Experimental Procedures"). All the other CR4-CR5 mTR mutants accumulated to the predicted size in the cell indicating that the mutated regions do not play a fundamental role in mTR processing. Complete deletion of the CR4-CR5 domain in mutants Delta 199-294 and Delta 172-314 accumulated to 16 and 30% of wild type levels, respectively (Fig. 2, a-c; Table I). Deletion of nonconserved loop L6 outside the CR4-CR5 domain in mutant Delta 220-241 also resulted in stable mTR accumulation (Fig. 2a; Table I).

The decreased RNA accumulation of the large deletion mutants in the pseudoknot and CR4-CR5 domains is likely to be due to an altered secondary structure of these mutant mTRs as a consequence of the big size of the deletion; in this regard, none of the point mutations and small deletions in these regions showed decreased accumulation as compared with wild type mTR.

The BoxH/ACA and CR7 Domains-- Most mutations affecting any of these two domains resulted in undetectable mTR in the cell 48 h after transfection, indicating that they contain essential regions for mTR stability in vivo. In particular, mTR mutants Delta 328-389 (BoxH/ACA and CR7) and Delta 357-364 (CR7), and both point mutants in the box H and box ACA conserved sequences, resulted in no detectable mTR accumulation in the cell (Fig. 2, a and b; Table I). Interestingly, not all mutations in these domains abolished mTR accumulation; mutants Delta 106-185 and Delta 172-314 that disrupt helix P4 in the BoxH/ACA domain accumulated at the predicted size and showed 41 and 30% of wild type mTR levels, respectively (Fig. 2, a and c; Table I). Similarly, deletion of four nonconserved J7b/8a unpaired nucleotides between conserved BoxH/ACA and CR7 domains in mutant Delta 341-345 resulted in stable accumulation (70% of wild type levels) (Fig. 2a; Table I). These results indicate that the length of the J7b/8a nonconserved hinge region between the two conserved domains as well as helix P4 are not important for mTR stability in vivo.

In Vivo Reconstitution of Telomerase Activity-- To identify mTR regions important for reconstitution of telomerase activity in vivo, extracts were prepared 48 h after transfection and were tested for telomerase activity using the TRAP assay (see "Experimental Procedures"). mTR-/- cells transiently transfected with wild type mTR showed an efficient reconstitution of telomerase activity in vivo (>50% of the parental wild type cell line activity). This activity was considered 100% telomerase activity reconstitution when compared with the different mTR mutants (see Blasco et al. (31) and mTR in Fig. 3, a-e; Table I). No reconstitution of telomerase activity was observed when the cells were transiently transfected with an empty vector (Fig. 3, a and e). To rule out that lack of telomerase reconstitution could be due to absence of mTERT accumulation in the cell, we did Western blot analysis of nuclear extracts from cells transfected with the different mTR versions by using the anti-telomerase-specific antibody K370 (10). In all cases, except in Box H mutant, similar levels of mTERT protein were detected (Fig. 4).



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Fig. 3.   In vivo reconstitution of telomerase activity. a-e, telomerase activity in wild type (mTR+/+) and telomerase-deficient KO3 p23 (mTR-/-) cells after transfection with wild type or mutant mTR constructs. The TRAP results are shown for illustrative purposes; some of the films shown are overexposed to allow the visualization of TRAP activity by mutants that are severely affected. Quantification was done using nonsaturated TRAP reactions and is shown in Table I. At 48 h after transfection, S-100 extracts were prepared and assayed for telomerase activity. All extracts were pretreated (+) or not (-) with RNase before being subjected to the telomerase assay. Protein concentration refers to total protein in the telomerase assay. Transfection with a plasmid containing the wild type mTR gene under its own promoter sequences was used as positive control for reconstitution of telomerase activity. Transfection with an empty vector (Bluescript) was used as negative control.



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Fig. 4.   mTERT protein expression after transfection of the different mTR mutations. Western blot analysis of total extracts from the indicated transfected cells using K-370 antibody. HeLa is a human cell line expressing high levels of human TERT (the mobility is slightly lower due to the fact that human TERT is larger than murine TERT). e.v, empty vector.

The Pseudoknot Domain-- All mutants disrupting the pseudoknot interaction (highly conserved helix P3) affected in vivo reconstitution of telomerase activity. In particular, mutants Delta 23-102, Delta 56-73, and Delta 120-143, all of which disrupt the P3 interaction, showed 10, 15, and 10% of wild type telomerase activity, respectively (Fig. 3, a and b; Table I). In contrast, mutant Delta 31-36, which eliminates the unpaired mammalian-specific J2a.1/2a sequence but does not affect the pseudoknot interaction, showed 130% of the wild type mTR telomerase activity (Fig. 3c), consistent with the fact that the J2a.1/2a sequence is not conserved (1). Deletion of four unpaired nucleotides in mutant Delta 90-94 decreased telomerase activity to 14% (Fig. 3c; Table I).

It has been previously shown in ciliates that the pseudoknot topology rather than the sequence is relevant for telomerase activity (36). To test whether a similar situation is true for vertebrate telomerase RNA pseudoknot, we generated 9 different substitution mutants. Mutant 65GCUGAC70 to 65GUCAGC70, which partially disrupted the ability of helix P3 to base pair but did not alter the size of the RNA, showed 20% of the wild type telomerase activity (Fig. 3b; Table I). Mutant 139GUCAGC144 to 139GCUGAC144, which partially disrupts helix P3, showed 22% of the wild type activity (Fig. 3b; Table I). A double substitution mutant 65GCUGAC70 to 65GUCAGC70/139GUCAGC144 to 139GCUGAC144 which fully restores the base pairing ability in helix P3 showed 81% of the wild type activity (Fig. 3b; Table I). We tried to confirm the above results by constructing new substitution mutants that completely disrupt the P3 helix. Mutant 65GCUGACUU72 to 65AAGUCAGC72 showed 30% of the wild type telomerase activity (Fig. 3e; Table I). Mutant 136AACGUCAGC144 to 136GCUGACGUU144 showed 15% of the wild type activity (Fig. 3e; Table I). However, a double substitution mutant 65GCUGACUU72 to 65AAGUCAGC72/136AACGUCAGC144 to 136GCUGACGUU144 showed 18% of wild type activity (Fig. 3e; Table I). All together, these results indicate that, in contrast to ciliate pseudoknot, in vertebrate telomerase RNA both pseudoknot topology and sequence are important for reconstitution of telomerase activity. We do not know if the vertebrate pseudoknot interacts with TERT, as has been shown for the ciliate pseudoknot (36); however, the fact that some of the mutants in this domain inhibit wild type telomerase activity suggests that it does (see below).

Three more mutants in the pseudoknot domain were tested that alter two sequences that are not base-paired in the mTR structure. Mutant 57UUUUU61 to 57AAAAA61 in the J2b/3 region showed 24% of wild type reconstitution activity, indicating that the identity of these residues is important for wild type reconstitution of telomerase activity in agreement with the fact that these residues are highly conserved in all vertebrates (Fig. 3b; Table I) (1). Mutant 129AAAAA133 to 129UUUUU133, which alters a nonconserved sequence in the J2a/3 region, showed 45% of wild type telomerase activity (Fig. 3b; Table I). A double substitution mutant 57UUUUU61 to 57AAAAA61/129AAAAA133 to 129UUUUU133 showed 13% of the wild type reconstitution activity (Fig. 3b; Table I), ruling out an interaction between 57UUUUU61 and 129AAAAA133 sequences, as predicted from the mTR structure (1).

Deletion of mTR unpaired regions in mutant Delta 154-167 reduces the distance between the pseudoknot domain and the CR4-CR5 and BoxH/ACA domains. This mutant showed 100% of wild type telomerase activity (Table I; Fig. 3b), indicating that the distance between these structural domains is not essential for reconstitution of telomerase activity, in agreement with the variable length of this region in vertebrate telomerase RNAs (1).

Mutant Delta 106-185, which removes J2a/3 sequences and disrupts the P3 helix in the pseudoknot domain, and the P4 helix in the BoxH/ACA domain showed 20% of the wild type mTR telomerase activity (Fig. 3a; Table I).

The CR4-CR5 Domain-- In contrast to pseudoknot domain deletion, full deletion of the CR4-CR5 domain in mutants Delta 199-294 and Delta 172-314 resulted in complete absence of telomerase reconstitution in vivo (Fig. 3, a and c), indicating that this domain contains essential regions for in vivo reconstitution of telomerase activity. In particular, deletion of several highly conserved residues in the J6/5 unpaired region of the CR4-CR5 domain in mutant Delta 254-266 completely abolished the ability of mTR to reconstitute active telomerase complexes in vivo (Fig. 3, b and c; Table I). Absence of telomerase activity reconstitution by mutant Delta 254-266 could be due to the altered mobility shown by this mutant (see above). Deletion of loop L6 outside the CR4-CR5 domain in mutant Delta 220-241 reduced telomerase activity to 20% that of wild type (Fig. 3, b and c), indicating that this loop is important but not essential for telomerase activity, in agreement with the fact that is not conserved in vertebrates (1).

The BoxH/ACA and CR7 Domains-- Some of the mutations in BoxH/ACA and CR7 domains resulted in no accumulation of mTR in vivo (see above) and should result in the absence of active telomerase complexes in vivo. Deletion mutant Delta 328-389, which removes part of the highly conserved BoxH/ACA domain (but not the highly conserved H and ACA boxes) and the CR7 domain, resulted in lack of reconstitution of active telomerase complexes (Fig. 3a; Table I), in agreement with undetectable accumulation of this RNA in the cell (see above). Similarly, a triple mutation in the invariable A residues of box H (A319U/A321U/A324U) and a double mutation in the invariable A residues of the ACA box (A392U/A394U) in the Box/ACA domain also abolished telomerase activity reconstitution (Fig. 3d). Finally, elimination of L8 loop in the CR7 domain in mutant Delta 357-364, also resulted in lack of telomerase activity reconstitution (Fig. 3, b and c). Interestingly, elimination of four nucleotides of the junction between the BoxH/ACA and CR7 domains (J7b/8a) in mutant Delta 341-345 did not affect telomerase activity, this mutant showing 112% of the wild type telomerase activity (Fig. 3c; Table I), concurring with the fact that the length of this hinge region is not conserved in vertebrate TRs (1). Similarly, deletion of P4 helix in the Box/ACA domain in mutant Delta 106-185 did not result in lack of reconstitution of telomerase activity (see above).

In Vivo Inhibition of Wild Type Telomerase Activity with mTR Dominant Negative Mutants-- Several of the mTR mutants studied affected telomerase reconstitution in vivo without significantly affecting mTR stability in the cell. Furthermore, some of these mTR mutations consist of small deletions and, therefore, are unlikely to alter correct mTR folding. These mutant mTRs are good candidates to act as telomerase inhibitors when overexpressed in wild type cells. The following mTR mutations were tested as potential dominant negatives: mutants Delta 120-143 and Delta 56-73 in the pseudoknot domain and mutant Delta 220-241 in the CR4-CR5 domain, all of which resulted in 80-90% reduction of wild type mTR reconstitution activity. We also constructed and studied an mTR mutant that lacked the template region, mutant Delta 3-10 (see "Experimental Procedures"). To check the ability of the different mTR mutations to inhibit wild type telomerase activity, we transfected a wild type 3T3 cell line (parental cells in Fig. 5) (31) with either an empty vector or with the different mutant mTR constructs, and in all cases the constructs contained a neomycin resistance gene for selection of stable clones (see "Experimental Procedures"). After selection with G418, several clones were isolated, and telomerase activity was assayed and quantified as described before. We considered that there was inhibition when telomerase activity was 30% or less that of the parental cell line. In the case of all mTR mutations assayed, but in none of the empty vector-transfected cells, we were able to isolate clones that showed inhibition of telomerase activity. Clones showing telomerase inhibition were 0% for the empty vector (0 of 8), 35% for Delta 3-10 mutant (5 of 14), 41% for Delta 56-73 mutant (5 of 12), 11.1% for Delta 120-143 mutant (1 of 9), and 45.5% for Delta 220-241 mutant (5 of 11). Fig. 5 shows telomerase activity quantification of mutant mTR-stable clones as compared with cells transfected with the empty vector or as compared with parental wild type cells (Fig. 5; see parental cells and empty vector lanes). These results show that mutations in the template, pseudoknot, and CR4-CR5 domains render mTR molecules that inhibit telomerase activity when expressed in wild type cells, suggesting that they act as dominant negatives. Furthermore, these results suggest that these regions are not essential for interaction with the mTERT subunit of telomerase.



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Fig. 5.   Telomerase activity inhibition in stable clones transfected with mTR dominant negative mutants. Telomerase activity in stable clones of wild type cells transfected with four different mutant mTR constructs and with the corresponding empty vector. Telomerase activity in the different clones was quantified as described above and represented in the graph relative to the telomerase activity shown by the parental cell line (normalized to 100%, parental cells). Six different empty vector-stable clones are shown (EV 1-6), all of them having similar or higher telomerase activity levels than the parental cell line. Five different mutant template-stable clones are shown (Delta 3-10 (4), Delta 3-10 (6), Delta 3-10 (10), Delta 3-10 (12), and Delta 3-10 (14)), all of them having 30% or less of the parental cell telomerase activity. Six different mutant pseudoknot-stable clones are shown (Delta 56-73 (1), Delta 56-73 (3), Delta 56-73 (4), Delta 56-73 (6), Delta 56-73 (9), and Delta 120-143 (5)), all of them having 30% or less of the parental cell telomerase activity. Five different mutant CR4-CR5-stable clones are shown (Delta 220-241 (4), Delta 220-241 (5), Delta 220-241 (8), Delta 220-241 (9), and Delta 220-241 (10)), all of them having 30% or less of the parental cell telomerase activity.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented here show that mutations in any of the four conserved mTR domains affect in vivo reconstitution of active telomerase complexes, suggesting that the full-length mTR molecule is important for in vivo formation of active telomerase complexes. In particular, our structure-function analysis supports a model in which vertebrate telomerase RNAs have two main functional domains as follows: (i) a domain that is essential for mTR stability, including the conserved BoxH/ACA and CR7 regions (highlighted in yellow in Fig. 6), and (ii) a domain that is important for activity and that includes the conserved pseudoknot and CR4-CR5 regions (highlighted in blue in Fig. 6). A previous report showed that hTR nucleotides from nucleotides 211 to 241, (including CR4-CR5, BoxH/ACA, and CR7 domains) were important for hTR accumulation and processing in the cell (26). In this regard, we find that regions within the BoxH/ACA and CR7 domains, but not in the CR4-CR5 domain, are essential for mTR accumulation in vivo. The BoxH/ACA domain has been also shown to be important for stability in small nucleolar RNAs (37, 38). Importantly, mutants affecting helix P4 of BoxH/ACA domain and the hinge region between BoxH/ACA and CR7 domains do not abolish mTR accumulation in the cell and are not highlighted in yellow in Fig. 6. Our results also suggest that the CR4-CR5 domain, formerly included in the "BoxH/ACA region" by Mitchell et al. (26), contains a highly conserved region that seems important for mTR processing (highlighted in green in Fig. 6).



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Fig. 6.   Summary of in vivo structure-function analysis in the mouse telomerase RNA component. Blue, regions important for in vivo reconstitution of telomerase activity; yellow, regions important for mTR accumulation in the cell; green, region that when deleted affects mTR processing; red, regions that when deleted act as dominant negatives of wild type telomerase activity.

As mentioned previously, the mTR domains important for reconstitution of telomerase activity include the pseudoknot and CR4-CR5 domains and are highlighted in blue in Fig. 6. Furthermore, the fact that mutants in these regions are able to inhibit wild type telomerase activity when overexpressed in vivo suggests that mutations in these regions do not abolish binding to mTERT. It is important to notice the fact that none of the pseudoknot mutants resulted in complete absence of telomerase activity reconstitution, suggesting that the pseudoknot domain is not essential for in vivo reconstitution of telomerase activity. In contrast, analysis of mutants in the CR4-CR5 domain suggested an essential role of CR4-CR5 domain in reconstitution of telomerase activity. This coincides with a previous report (26) that suggested that nucleotides 211-241 in hTR (including the CR4-CR5 domain) contained regions that were important for telomerase activity. In contrast, in vitro reconstitution studies yielded different results regarding the minimal hTR region required for telomerase activity. The studies by Autexier et al. (28), which mapped the minimal functional region of hTR to nucleotides 44-203 using a reconstitution system with Micrococcus nuclease-treated, partially purified human 293 cells, and that by Beattie et al. (29), which mapped the minimal hTR region to nucleotides 10-154 using in vitro produced hTR and hTERT, do not coincide with the in vivo reconstitution results that show that the conserved CR4-CR5 domain is essential for telomerase activity. The results we obtained with the in vivo reconstitution system are coincidental with those by Tesmer et al. (30) that described that hTR nucleotides 1-325 (including the CR4-CR5 domain) but not 1-300 were sufficient to reconstitute wild type telomerase activity, again suggesting that the CR4-CR5 domain is important for telomerase activity.

It is important to notice that mutants in mTR regions that are not conserved in other vertebrate telomerase RNAs were less critical for both mTR activity and stability.

Structure-function studies in mammalian telomerase RNAs are a necessary step for a rational design of telomerase inhibitors. In this regard, we identify here several short sequences in the template, pseudoknot, and CR4-CR5 conserved domains that when mutated act as dominant negatives. These three mTR unpaired regions in the template, pseudoknot, and CR4-CR5 domains are good candidates for antisense inhibition of telomerase.


    ACKNOWLEDGEMENTS

We thank Carol Greider for sharing the vertebrate telomerase RNA secondary structure before publication and for providing Fig. 1, and Manuel Serrano and Cathy Mark for critical reading of the manuscript. The Department of Immunology and Oncology was founded and is supported by the Spanish Research Council (CSIC) and Amersham Pharmacia Biotech and The Upjohn Co.


    Addendum

While preparing this manuscript for publication, a paper by Mitchell and Collins (39) describing a mutational analysis of the hTR BoxH/ACA conserved region was published. Our results are in agreement with those presented by these authors and, in particular, with the finding that the CR4-CR5 domain contains residues essential for telomerase activity.


    FOOTNOTES

* This work was supported in part by the Swiss Bridge Award 2000, by Grant PM97-0133 from the Ministry of Science and Technology of Spain, by Grant 08.1/0030/98 from the regional Government of Madrid, Spain, by Grants "TACID," "TELORAD," and "SUS GENES IN RAD CAR" from the European Union, and by the Department of Immunology and Oncology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a predoctoral fellowship from the Spanish Ministry of Education and Culture.

§ To whom correspondence should be addressed. E-mail: mblasco@cnb.uam.es.

Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008419200


    ABBREVIATIONS

The abbreviations used are: TERT, telomerase reverse transcriptase; TR, telomerase RNA; kb, kilobase pairs; PCR, polymerase chain reaction.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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