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
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ABSTRACT |
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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 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 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:
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:
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 Transfection of mTR 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).
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).
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
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.
The Pseudoknot and CR4-CR5 Domains--
mTR deletions
Interestingly, deletion of several conserved nucleotides of the J6/5
region in mutant
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
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 The Pseudoknot Domain--
All mutants disrupting the pseudoknot
interaction (highly conserved helix P3) affected in vivo
reconstitution of telomerase activity. In particular, mutants
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
Mutant The CR4-CR5 Domain--
In contrast to pseudoknot domain deletion,
full deletion of the CR4-CR5 domain in mutants 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 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 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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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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
3-10,
5'GGGTATTTAAGGTCGAGGGCGGCTAGGCCTCGGCACGATTTTCATTAGCTGTGGGTTCTGG3';
23-102, 5'CACCTAACCCTGATTTTCATTAGCGGGCCACCGCGCGTTCCCG3';
106-185, 5'GAAAGTCCAGACCTGCAGCGGCGCCCAGTCCCGTACCCGCCTACAGGCCG3';
199-294, 5'GCCGGGCCGCCCAGTCCCGTCCAGGCCGGGCGAGCGCCGCG3';
328-389,
5'GCGCCGCGAGGACAGGAATGTCACAACCCCCATTCCCGCTG3';
56-73,
5'GGTCTTTTGTTCTCCTCCGCCCGCTGCAGCGGGCCAGGAAAGTCCAG3';
120-143, 5'CCAGACCTGCAGCGGGCCACCGCGCGTTCCGCAGGAGCTCCAGGTTCG3';
154-167, 5'CCTCAAAAACAAACGTCAGCGCAGGAGCTAGCTCCGCGGCGCCGGGCCGCCCAGTCCCG3';
220-241,
5'CCGTACCCGCCTACAGGCCGCGGCCGGCCCTGCCGCCGCGAAGAGCTCGCCTCTGTCAGC3';
254-266, 5'GGGTCTTAGGACTCCGCTGCCGCCGCGAGTCAGCCGCGGGGCGCCGGGGGCTG3';
357-364, 5'GGTCCCCGTGTTCGGTGTCTTACCGGGAAGTGCACCCGGAACTCGGTTC3';
341-345, 5'GGACAGGAATGGAACTGGTCCCCGGGTGTCTTACCTGAGCTGTGG3';
31-3, 5'CCCTGATTTTCATTAGCTGTGGGTCTTTTGTTCTCCGCCCGCTGTTTTTC3';
90-94, 5'GCTGACTTTCCAGCGGGCCAGGAAAGACCTGCAGCGGGCCACCGCGTTCCCGAGCCTC3'; substitution 57-61 U
A,
5'GGTCTTTTGTTCTCCGCCCGCTGAAAAACTCGCTGACTTCCAGCGGGCCAGGAAAGTCC3'; substitution 129-133 A
U,
5'CACCGCGCGTTCCCGAGCCTCTTTTTCAAACGCAGCGCAGGAGCTCC3'; substitution
65-70 GCUGAC
GUCAGC,
5'GGTCTTTTGTTCTCCGCCCCTGTTTTTCTCGTCAGCTTCCAGCGGGCCAGGAAAGTCCAG3'; substitution 139-144 GUCAGC
GCUGAC,
5'-GTTCCCGAGCCTCAAAAACAAACCTGACGCAGGAGCTCCAGGTTC3'; substitution 71-74 UUCC
GGAA,
5'GGTCTTTTGTTCTCCGCCCGCTGTTTTTCTCGCTGACGGAAAGCGGGCCAGGAAAGTCCAG3'; substitution 71-74 + 84-87 UUCC
GGAA + GGAA
UUCC,
5'GTTTTTCTCGCTGACGGAAAGCGGGCCATTCCAGTCCAGACCTG3'; box H mutant
A319U/ A321U/A324U,
5'CAGGCCGGGCGAGCGCCGCAAGGTCTGGTATGGAACTGGTCCCCGTG3'; box ACA mutant
A392U/A394U,
5'GCACCCGGAACTCGGTTCTCTCTACCCCCATTCCCGCTGGGGAAATGCC3'.
172-314, forward, 5'CCAGGTTCGCCGGGAGCTCCGAGGACAGGAATGGAACTGG3';
172-314, reverse, 5'CCAGTTCCATTCCTGTCCTCGGAGCTCCCGGCGAACCTGG3';
substitution 65-72 GCUGACUU
AAGUCAGC, forward,
5'GGTCTTTTGTTCTCCGCCCGCTGTTTTTCTCCGACTGAACCAGCGGGCCAGGAAAGTCCAG3'; substitution 65-72 GCUGACUU
AAGUCAGC, reverse,
5'CTGGACTTTCCTGGCCCGCAGGTTCAGTCGGAGAAAAACAGCGGGCGGAGAACAAAAGACC3'; substitution 136-144 AACGUCAGC
GCUGACGUU, forward,
5'CGTTCCCGAGCCTCAAAAACATTCCAGTCGGCAGGAGCTCCAGGTTCG3'; substitution
136-144 AACGUCAGC
GCUGACGUU, reverse,
5'CGAACCTGGAGCTCCTGCCGACTGGAATGTTTTTGAGGCTCGGGAACG3'.
, 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.
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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
-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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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).
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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
254-266.
Summary of stability and activity of the different mTR versions
23-102,
31-36, and
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
120-143 and
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 (
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
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).
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
199-294 and
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
220-241 also resulted in stable mTR accumulation (Fig.
2a; Table I).
328-389 (BoxH/ACA and CR7) and
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
106-185 and
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
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.
/
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.
23-102,
56-73, and
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
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
90-94 decreased telomerase activity to 14%
(Fig. 3c; Table I).
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).
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).
199-294 and
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
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
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
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).
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
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
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
106-185 did not result in lack of reconstitution
of telomerase activity (see above).
120-143 and
56-73 in the pseudoknot domain and mutant
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
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
3-10 mutant (5 of 14), 41% for
56-73 mutant (5 of 12), 11.1%
for
120-143 mutant (1 of 9), and 45.5% for
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
( 3-10 (4),
3-10 (6),
3-10 (10),
3-10 (12), and
3-10 (14)), all of them having 30% or less
of the parental cell telomerase activity. Six different mutant
pseudoknot-stable clones are shown (
56-73 (1),
56-73 (3),
56-73 (4),
56-73 (6),
56-73 (9), and
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 (
220-241 (4),
220-241 (5),
220-241 (8),
220-241 (9), and
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
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[in a new window]
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.
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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.
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are: TERT, telomerase reverse transcriptase; TR, telomerase RNA; kb, kilobase pairs; PCR, polymerase chain reaction.
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