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INTRODUCTION |
Telomerase is a ribonucleoprotein
(RNP)1 that is responsible
for maintaining the terminal repeats of telomeres in most organisms (1). It acts as an unusual reverse transcriptase, using a small segment
of an integral RNA component as template for the synthesis of the
dG-rich strand of telomeres (2).
Telomerase activity has been characterized from a wide range of
organisms and genes encoding both the RNA and protein components of the
enzyme complex identified (for reviews see Refs. 3 and 4). Telomerase
RNAs found in ciliated protozoa, in addition to having a short
templating region, share a common secondary structure. Telomerase RNAs
from yeast and mammals are considerably larger, and within each group
conserved structural elements can be identified based on phylogenetic
and mutational analysis (5, 6). The catalytic reverse transcriptase
protein subunit (TERT), first purified from Euplotes
aediculatus as p123, was found to be homologous to Est2p, a
protein from Saccharomyces cerevisiae required for telomere
maintenance (7-9). Both proteins possess reverse transcriptase
(RT)-like motifs, alterations in which render telomerase inactive
both in vitro and in vivo. Subsequently,
homologues of TERT were identified in a phylogenetically diverse group
of organisms (10-17). Because co-expression of TERT and telomerase RNA
in rabbit reticulocyte lysates suffices to reconstitute enzyme activity
(18, 19), these two subunits probably constitute the core of the enzyme
complex. Quite a few telomerase-associated polypeptides have been
identified using either biochemical or genetic tools. Studies from
several laboratories (20-24) suggest that these factors may
participate in telomerase assembly, catalytic function, or regulation.
The RT-like motifs are located in the C-terminal region of cloned
TERTs. Extensive mutational analysis of these motifs in TERTs supports
an overall conservation of basic catalytic mechanisms between
telomerase and conventional RTs. For example, the TERT analogues of RT
residues essential for catalysis are absolutely required for telomerase
activity and telomere maintenance (9, 18, 25-27). Conserved residues
shown previously (28-30) to modulate RT processivity have been found
to be important determinants of telomerase processivity as well. In
addition, the same tyrosine residue in conserved motif A allows both
TERTs and RTs to discriminate against incorporating ribonucleotides
(31). However, some other crucial RT residues (e.g. a Gln in
motif B') appear to be less important or even dispensable for
telomerase catalysis (9). Taken together, these results suggest that
despite the high degree of sequence divergence (<20% sequence
identity), the RT domains in both classes of proteins are
mechanistically quite similar.
Detailed sequence analysis of nine cloned TERTs revealed, in addition
to the RT motifs, four telomerase-specific motifs (named GQ, CP, QFP,
and T) positioned N-terminal to the RT region (32). The functions of
selected TERT N-terminal residues have been analyzed by reconstituting
mutated TERT protein with telomerase RNA either in vitro
(Tetrahymena and human) or in vivo (S. cerevisiae, Schizosaccharomyces pombe, and human) (18,
27, 31-33). In addition to confirming the functional importance of
many of the conserved residues, the results from Tetrahymena
indicate that the CP and T motifs may be required for TERT binding to
telomerase RNA in vitro (33, 34).
We have initiated a detailed structure-function analysis of the
N-terminal region of S. cerevisiae TERT (Est2p). In an
earlier report (32), we showed that the most N-terminal GQ motif is required for telomere maintenance and, in some cases, for telomerase activity in vitro. Overexpression of some of the mutants
resulted in telomere shortening, implying that telomerase
catalytic function, rather than RNP formation, was impaired by the
mutations. We now report an analysis of the remaining three N-terminal
motifs using an extensive panel of mutations. Our results confirm the
functional importance of the CP, QFP, and T motifs in both telomere
maintenance and telomerase activity. In addition, for mutations in
these motifs, the extent of telomerase activity loss correlates with
the extent of reduction in the amount of TERT protein and
TERT-associated TLC1 RNA. Overexpression of the mutant proteins does
not result in telomere shortening, implying that assembly rather than
catalytic function was affected. This notion was further supported by a comparison between the efficiency of RNP formation in the wild type and
the overexpression strains. Taken together, our results imply that
three of the four N-terminal motifs in TERTs are required for efficient
RNP formation in vivo but not for the telomere extension function of telomerase. We also show that the majority of
telomerase-associated TLC1 RNA has a more upstream 3' end than reported
previously, consistent with additional processing events during RNP maturation.
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MATERIALS AND METHODS |
Yeast Strains and Plasmids--
The construction of an
est2-
strain harboring the pSE-Est2-C874 plasmid
(containing a protein A-tagged EST2 gene) has been described
(32). This fully functional Est2p is designated wild type telomerase
throughout the text. The construction of the protein A-tagged C745
mutant has also been described (29). All substitution mutations in the
CP, QFP, and T motifs of EST2 were generated by using the
Quick-Change protocol (Stratagene), appropriate primer oligonucleotides, and pSE-Est2-C874 as template. All point mutations were confirmed by sequencing. The oligodeoxynucleotide primers used for
mutagenesis were purchased from Sigma-Genosys and purified by
denaturing gel electrophoresis prior to use. For overexpression of TERT
mutants, a vector containing the triose-phosphate isomerase promoter
(pYX232, Ingenious Inc.) was utilized. The NcoI site within
the polylinker of pYX232 was converted to an NdeI site, and
the NdeI-SalI fragments from the pSE-Est2-C874
series of plasmids were inserted between the NdeI and
SalI site of the resulting vector (32).
Comparative Sequence Analysis--
All sequences used in
comparative analysis were obtained from NCBI website at
www.ncbi.nlm.nih.gov. The final alignment was based on manual
adjustment of a hidden Markov model as described previously (32).
Analysis of Telomere Length--
The est2-
and
W303 strain were transformed with the pSE and pYX series of plasmids,
respectively. Independent clones were re-streaked twice on plates.
Chromosomal DNAs were then isolated using the "Smash and Grab"
protocol, digested with PstI, and electrophoretically separated on a 0.9% agarose gel. Following capillary transfer to nylon
membranes, telomere-containing fragments were detected by hybridization
with a 32P-labeled poly(dG-dT) probe (32).
Purification of and Assay for Yeast Telomerase--
Whole cell
extracts, DEAE fractions, and IgG-Sepharose-purified telomerase were
prepared as described previously (29, 32, 35, 36). Each primer
extension assay was carried out using 20 µl of IgG-Sepharose
pretreated with 4 mg of protein extract and was initiated by the
addition of a 15-µl mixture containing 100 mM Tris·HCl,
pH 8.0, 4 mM magnesium chloride, 2 mM
dithiothreitol, 2 mM spermidine, primer
oligodeoxynucleotides, and varying combinations of labeled and
unlabeled dGTP and dTTP. Primer extension products were processed and
analyzed by gel electrophoresis as described previously (36, 37). The
oligodeoxynucleotide primers used for telomerase assays were purchased
from Sigma-Genosys and purified by denaturing gel electrophoresis prior
to use. The primers have the following sequences: TEL15,
TGTGTGGTGTGTGGG; TEL66, TAGGGTAGTAGTAGGG.
Protein and RNA Analysis--
The levels of protein A-tagged
yeast TERT in cells extracts were determined by Western blotting as
described previously (30). RNase protection studies were carried out as
follows. A PCR fragment that spans nucleotide 1-1301 of the
TLC1 gene (38) was generated and cloned between the
BamHI and EcoRV site of pBluescript II KS+ to
give pBS-TLC1. For the synthesis of labeled antisense probe, pBS-TLC1
was linearized by digestion with HinfI and transcribed with
T3 RNA polymerase in the presence of 12 µM
[
-32P]GTP as described (39). For the synthesis of the
1-1300 and 1-917 sense transcripts, the same plasmid was digested
with XhoI and AflII, respectively, and
transcribed with T7 RNA polymerase. For the protection assay, total
RNAs from DEAE fractions or in vitro transcription reactions
were combined with the probe (100,000 cpm), and the mixtures were
precipitated with ethanol. The RNAs were then hybridized in 80%
formamide, digested successively with RNase T1, RNase A, and proteinase
K, and analyzed with gel electrophoresis as described (40).
High Resolution Mapping of the TLC1 RNA 3' End--
Total RNAs
from DEAE fractions or from in vitro transcription reactions
were isolated by proteinase K digestion, phenol/chloroform/isoamyl alcohol extraction, and ethanol precipitation. The RNAs were then polyadenylated using yeast poly(A) polymerase and reverse
transcribed using a poly(dT)-containing primer
(CCGGAATTCTTTTTTTTTTTTTTTTTT) (41). cDNAs spanning the 3' end of
TLC1 RNA were amplified by PCR using a TLC1-internal primer
(CGGGATCCGATCAGTAACTGAACAATGAC) and the same poly(dT) primer.
Following gel purification and restriction enzyme cleavage, the
fragments were cloned between the EcoRI and BamHI
site of pBluescript II KS+, and the TLC1-poly(A) junctions were
determined by sequencing.
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RESULTS |
Previously Identified N-terminal Motifs of TERT Are Conserved in
Newly Discovered Homologues--
Through the use of a hidden Markov
model, we had earlier reported the identification of four conserved
motifs in the N-terminal extension of TERTs from ciliates, fungi,
mammals, and a plant (32). Subsequently, sequences for 11 additional
TERTs were reported, including those from Cryptosporidium
parvum, Encephalitozoon cuniculi, Giardia
lamblia, Mesocricetus auratus, Moneuplotes
crassus, Oryza sativa, Paramecium caudatum,
Plasmodium yoelii, Plasmodium falciparum, Xenopus laevis, and Caenorhabditis
elegans (42-50). Some of the newly discovered homologues manifest
a surprising degree of evolutionary divergence. For example, the TERTs
from P. falciparum and P. yoelii are more than
twice the size of many other proteins of this family. Nevertheless, a
realignment of the N-terminal extension suggests that, with the
exception of CeTERT, the four N-terminal motifs are universally
conserved (Fig. 1). Close inspection of
the new alignment revealed the following two interesting features.
First, even though there are very few absolutely conserved residues, many positions appear to tolerate only conservative substitutions. A
great majority of these conserved positions favor hydrophobic or
aromatic residues, which may correspond to key positions in the core of
a domain or surface locations crucial for interactions with other
domains or molecules. Second, the proposed linker region between the GQ
and CP motif is even more variable in length than previously suspected,
ranging in size from 19 residues to >500. Interestingly, the smallest
TERT (E. cuniculi) also possesses the smallest linker.

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Fig. 1.
Hidden Markov model-generated multiple
sequence alignment for the N-terminal region of known and predicted
telomerase reverse transcriptases (TERT). Conserved residues are
highlighted, and hydrophobic positions are boxed.
Triangles indicate the locations of the S. cerevisiae mutations examined in this work. Numbers
indicate the number of residues not depicted explicitly. The sequences
and their data base codes are as follows: Tt_TERT,
Tetrahymena thermophila TERT [TERT_TETTH];
Ot_TERT, Oxytricha trifallax TERT [TERT_OXYTR];
Ea_TERT, E. aediculatus TERT [TERT_EUPAE];
Mc_TERT, M. crassus TERT [AAM95622];
Pc_TERT, P. caudatum TERT [Q9GRC5];
Sc_Est2p, S. cerevisiae TERT [TERT_YEAST];
Sp_TRT1, S. pombe TERT [TERT_SCHPO];
Ca_TERT1, Candida albicans TERT 1 [Q9P8T3];
Xl_TERT, X. laevis TERT [Q9DE32];
Mm_TERT, Mus musculus TERT [TERT_MOUSE];
Ma_TERT, M. auratus TERT [Q9QXZ4];
Hs_TERT, Homo sapiens TERT [TERT_HUMAN];
Os_TERT, O. sativa TERT [Q9AU13];
At_TERT, Arabidopsis thaliana TERT [Q9SE99];
Gl_TERT, G. lamblia TERT [Q9NCP5];
Pf_ORF, P. falciparum 3D7 hypothetical protein
PF13_0080 [NP_705050]; Py_TERT, P. yoelii TERT
[EAA16235]; Cp_TERT, C. parvum TERT
[AAK60396]; and Ec_TERT, E. cuniculi TERT
[NP_596954].
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The CP, QFP, and T Motifs of Yeast TERT Are Required for Telomere
Maintenance and Telomerase Enzyme Activity--
To extend our earlier
functional analysis of the GQ motif, we created a series of
substitution mutants of yeast TERT with alterations in the CP, QFP, and
T motifs, and we tested their abilities to support telomere
maintenance. A total of 18 mutants with substitution in
conserved residues (SCR mutants) were
generated, each with one or two residues (and in one case, three
residues) changed to alanines: IL256, CP261, LE264, F286, IL290,
LLP294, MF299, LL316, LL330, DF338, WL341, W367, LI372, II376, FF380, T384, Y394, and W400. Each mutant is designated by the identity and
location of the altered amino acid residue. Where more than one residue
is mutated, the number specifies the position of the first residue of
the pair or triplet. For comparison, a mutant with substitutions in
non-conserved residues was also made (GK307). Each mutant was tagged at
the C terminus with tandem copies of the IgG-binding domain of proteins
A, placed on a centromeric plasmid, and used to transform a yeast
strain whose chromosomal EST2 (yeast TERT) gene has been
disrupted. A similarly tagged wild type EST2 gene was
analyzed in parallel as the control.
All of the strains were first tested for telomere maintenance defects.
As predicted and shown in Fig. 2, the
majority of SCR mutants exhibited varying degrees of telomere
shortening. Based on the degree of shortening, the mutant phenotypes
were classified as mild (CP261, LE264, GK307, W367, T384, and W400;
less than 50 bp shorter than wild type), intermediate (F286, IL290,
MF299, LL316, DF338, WL341, LI372, and Y394; 50-200 bp shorter than
wild type), or severe (LLP294, LL330, II376, and FF380; more than 200 bp shorter than wild type). The four strains with severe telomere shortening (LLP294, LL330, II376, and FF380) also exhibited reduced growth on agar plates as evidenced by reduced colony size (data not
shown). In addition, these strains display evidence for amplification of the subtelomeric Y' fragment (indicated by filled circles
in Fig. 2), which has been shown previously (51) to correlate with senescence and the generation of type I survivors. As predicted, the
single non-SCR mutant (GK307) exhibited no defect in telomere maintenance. Surprisingly, five of the SCR mutants (CP261, LE264, W367,
T384, and W400) showed either normal telomeres or very mild shortening.
However, where it has been tested in other systems, the equivalent
residues appear also to be relatively unimportant for telomerase enzyme
activity or telomere maintenance (18, 33). We conclude that sequence
alignment in the CP, QFP, and T region is useful for identifying
functionally important TERT residues.

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Fig. 2.
Mutations in the CP, QFP, and T motifs impair
telomere maintenance. Telomere lengths were determined for strains
bearing wild type or mutated TERT. The identities of the mutants are
indicated at the top of the panel. The location of Y' type
telomeres is indicated by brackets. The amplified Y'
fragments that are often observed in senescent strains are marked by
filled circles.
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To determine whether the telomere maintenance defects can be explained
by telomerase activity loss or alteration, we precipitated protein
A-tagged telomerase from the wild type and mutant strains by adsorption
to IgG-Sepharose, and we subjected the Sepharose beads to primer
extension analysis. All of the mutant enzymes were tested side-by-side
with wild type telomerase using a 16-mer oligonucleotide with
non-canonical telomere repeats (TEL66) as the primer substrate (Fig.
3, A and B). In
most cases, comparisons were also made using a 15-nt primer with
canonical repeats (TEL15), which yielded very similar results (Fig.
3B, and data not shown). As shown in representative assays
and the summary plot (Fig. 3C), there is an excellent
correlation between the extent of telomere shortening and telomerase
activity reduction. Mutant strains with the most severe telomere length
defect contain essentially no telomerase activity (LLP294, LL330,
II376, and FF380). For mutants with intermediate telomere defects, the
reduction in telomerase activity ranges from ~65% for F286 (which
exhibits the least telomere length defect among the intermediate group)
to ~95% for DF338, WL341, LI372, and Y394 (which have the most
length defect in this group). Mutant strains with mild or no defect had
close to or more than 50% of the wild type level of telomerase
activity (CP261, LE264, GK307, W367, T384, and W400). None of the
mutants with detectable telomerase exhibited an obvious processivity
defect (Fig. 3, A and B, and data not shown).
Taken together, our results indicate that mutations in the CP, QFP, and
T motif impair simultaneously telomere maintenance and telomerase
activity but not telomerase processivity.

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Fig. 3.
Mutations in the CP, QFP, and T motifs lead
to loss of telomerase activity. A, telomerase from the wild
type and various mutant strains was isolated by IgG affinity
chromatography and tested in primer extension assays using TEL66 as the
primer, and [32P]dGTP and dTTP as the nucleotides. The
identities of the mutations are indicated at the top and the
location of the primer+3 (+3) product indicated by horizontal
lines. B, telomerase from the wild type and various
mutant strains was isolated by IgG affinity chromatography and tested
in primer extension assays using TEL66 or TEL15 as the primer, and
[32P]dGTP and dTTP as the nucleotides. The identities of
the mutations are indicated at the top and the location of
the primer+3 (+3) product indicated by horizontal lines. In
these assays, a labeled oligonucleotide was precipitated along with the
reaction products as a recovery control (arrows).
C, total DNA synthesis mediated by each mutant enzyme was
determined in assays such as those presented in part A and
B, normalized against that mediated by the wild type enzyme,
and the results plotted. The values are averages of two independent
assays.
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Mutations in the CP, QFP, and T Motifs Caused a Reduction in the
Amount of TERT Protein and TERT-associated TLC1 RNA--
To determine
the basis for the activity loss manifested by the SCR mutants, we
estimated the levels of TERT protein in wild type and mutant strains
using antibodies directed against the protein A tag. The levels of TERT
protein in the mutant strains were gauged by comparing the signal
obtained from a fixed amount of mutant extract with those from varying
amounts of wild type extract. As shown in Fig.
4, A and B, the
mutants with significant telomere length defects consistently
manifested a reduction in the amount of TERT protein. In addition, the
extent of functional loss correlates loosely with the degree of protein
reduction. For instance, the mutants with severe telomere shortening
all had undetectable levels of TERT protein (LLP294, LL330, II376, and
FF380), whereas those with intermediate telomere defects had measurable
levels (e.g. IL290, MF299, and LL316).

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Fig. 4.
Mutations in the CP, QFP, and T motifs lead
to reductions in the level of TERT protein. A, levels of
protein A-tagged TERT in the mutant strains were determined by Western
blotting using antibodies directed against protein A and compared with
that of the wild type strain. The identities of the mutants are
indicated at the top, and the relative amounts of extracts
loaded indicated at the bottom. The amount 1×
denotes 400 µg of total protein. B, the levels of protein
A-tagged TERT in the mutant strains were estimated visually based on
assays such as those presented in A and the results
summarized. The levels are scored as follows: , <12.5% of wild
type; +, 12.5-25% of wild type; ++, 25-50% of wild type; +++,
>50% of wild type. Most of the assays (specifically, the CP and QFP
mutants) were performed twice, with very similar results.
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The amount of TERT-associated TLC1 RNA was measured by IgG-Sepharose
adsorption of tagged telomerase followed by RNase protection analysis
(Fig. 5). In most experiments, a single
protected species can be detected (Fig. 5A). However, in
some experiments, an additional smaller band can be visualized,
presumably arising from excessive degradation of the hybrid by RNases
(Fig. 5B). Because both bands are due to TLC1 RNA
protection, both are included in the calculation of the amount of this
RNA. Comparison of the summary plots in Figs. 5C and
3C revealed an excellent correlation between the extent of
activity loss and the degree of reduction in the level of
TERT-associated TLC1 RNA. Mutants with close to wild type telomerase activity had high levels of TERT-associated RNA (CP261, LE264, GK307,
W367, T384, and W400), whereas those with severe defects had little or
no associated RNA (LLP294, LL330, II376, and FF380). These results
indicate that the loss of telomerase activity in the mutant strains may
be accounted for by the loss of TERT and TERT-associated TLC1 RNA.
Furthermore, analysis of mutants with appreciable activities suggests
that the telomerase complexes containing mutant proteins have specific
activities that are comparable with those containing wild type TERT.
For example, although the DF338, WL341, LI372, and Y394 mutant each
suffered a 90-95% reduction in activity, each also suffered a
90-95% reduction in TERT-associated RNA (Figs. 3C and
5C). The mutant proteins therefore appear to be
catalytically competent once associated with RNA.

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Fig. 5.
Mutations in the CP, QFP, and T motifs cause
reductions in the level of TERT-associated TLC1 RNA. A, the
levels of TERT-associated TLC1 RNA in the wild type and mutant strains
were determined by RNase protection assays. The identities of the
mutants are indicated at the top. As a control, an extract
was prepared from an untagged strain and subjected to the same
analysis. The position of the protected TLC1 fragment is indicated by
an arrow on the right. A nonspecific band, marked
by a diamond, can sometimes be seen in these assays.
B, the levels of TERT-associated TLC1 RNA in the wild type
and mutant strains were determined by RNase protection assays. The
identities of the mutants are indicated at the top. The
positions of two protected fragments are indicated by arrows
on the right. C, the levels of TERT-associated
TLC1 RNA in the mutant strains relative to the wild type strain were
determined by assays such as those shown in A and
B, and the average values from two experiments
plotted.
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Overexpression of the Mutant Proteins Does Not Result in Telomere
Shortening--
The concomitant reduction in the level of telomerase
activity, TERT protein, and TERT-associated TLC1 RNA suggests that
mutations in the CP, QFP, and T motifs affected RNP assembly rather
than the in vivo and in vitro activity of
telomerase. To explore this possibility, we overexpressed various
mutant proteins in a wild type strain background, and we analyzed the
lengths of telomeres in the resulting strains. If the mutant proteins
are defective in assembly, then they should not compete with endogenous
TERT for association with TLC1 RNA and should not act in a
dominant-negative fashion even when overexpressed. This was indeed
found to be the case. Five SCR mutants of TERT (LLP294,
MF299, LL330, II376, and FF380) were cloned downstream of the strong
and constitutive triose-phosphate isomerase promoter, and the resulting
plasmids were introduced into a reference strain containing a native
TERT gene (W303). Following two re-streaks (~50
generations), chromosomal DNAs were isolated from the clones and
analyzed for telomere length alteration (Fig.
6). As a control, a C-terminal truncation
mutant (C745), previously shown to be defective in telomerase activity
and processivity but not assembly, was tested in parallel (29). As
expected, the C745 mutant, when overexpressed, caused dramatic telomere shortening, by ~200 bp. In contrast, none of the SCR mutants (with alterations in the QFP and T motif) was able to act in a
dominant-negative fashion with regard to telomere maintenance. We
are led to conclude that the major deficiency engendered by the
mutations is in telomerase assembly rather than enzyme function.

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Fig. 6.
Overexpression of proteins with mutations in
the QFP and T motifs in a wild type strain background does not cause
telomere shortening. The yeast strain W303 was transformed with a
pYX232 plasmid overexpressing a wild type or a mutated TERT. After the
transformants were re-streaked twice, chromosomal DNAs were isolated
from the strains, digested with PstI, and analyzed for
telomere lengths. The identities of the overexpressed proteins in the
transformants are indicated at the top.
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The Efficiency of RNP Formation Is Reduced by the
Mutations--
In the non-overexpression strains analyzed in Figs.
2-5, the concurrent loss of TERT and TERT-associated RNA can be
explained as either a direct or indirect consequence of mutations on
protein stability. In the former scenario, the mutations might have
caused protein misfolding, leading to accelerated degradation. Fewer TERT molecules would then be available for association with TLC1 RNA.
In the latter scenario, the mutations disrupted protein-RNA interaction, resulting in less RNP formation. The unincorporated protein molecules were then preferentially degraded. To distinguish between these possibilities, we examined the efficiency of RNP formation in our overexpression strains. As described earlier, these
strains contain, in addition to the native TERT gene, a mutated TERT (tagged with protein A) that is under the
control of the strong triose-phosphate isomerase promoter. For
comparison, a yeast strain with a tagged wild type gene driven by the
native promoter was tested in parallel. As judged by the Western
analysis shown in Fig. 7 (top
panel), all of the overexpressed mutant proteins accumulated to
higher levels than the non-overexpressed wild type protein, which was
undetectable in this particular experiment. (The non-overexpressed wild
type protein can be detected when the developing time for the blot in
the color substrates is increased, as seen in Fig. 4A.)
Interestingly, the steady-state level of the LL330 mutant protein is
lower than the other mutant proteins, implying some additional defect
in protein stability. The levels of TLC1 RNA associated with mutant
proteins were measured by IgG-Sepharose adsorption of tagged telomerase
followed by RNase protection analysis. Of the mutant proteins, only
C745 co-precipitated an increased amount of TLC1 RNA (Fig. 7,
middle panel, compare lanes 1 and 7).
The other mutants, despite their increased expression, associated with
either comparable or reduced levels of RNA in comparison with the
non-overexpressed native protein (Fig. 7, middle panel, compare lanes 2-6 with lane 7). Furthermore, the
efficiency of RNA association correlates with the extent of functional
loss in vivo. For example, of the five overexpressed SCR
mutants, MF299 was able to associate with the highest amount of TLC1
RNA when overexpressed. This mutant was also the least impaired in
terms of its ability to maintain telomeres in the absence of wild type protein (Fig. 2). These results suggest that the major defect of the
mutant proteins is their reduced ability to associate with telomerase
RNA.

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Fig. 7.
Analysis of the efficiency of RNP assembly in
the overexpression strains. W303-derived strains that overexpress
various TERT mutants were analyzed for TERT protein levels (top
panel), TERT-associated TLC1 RNA (middle panel), and
TERT-associated telomerase activity (bottom panel) by
Western, RNase protection, and primer extension assays, respectively.
For comparison, a strain carrying a tagged wild type TERT
gene under the control of the native promoter was tested in parallel
(lane 7). The identities of the overexpressed TERT mutant
proteins are indicated at the top.
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Mapping of the 3' End of TLC1 RNA in Telomerase RNP--
In the
process of quantifying the amount of TERT-associated TLC1 RNA, we
noticed that the length of the probe protected by the RNA appears to be
shorter than that predicted by previous analysis. According to a
published report (41), the de-adenylated, mature RNA is thought to have
3' ends that cluster near position 1250. If this were true, then the
RNase protection assay should yield a protected fragment of ~150 nt.
(See Fig. 8A for a schematic illustration of the RNase protection assay.) However, our analysis of
telomerase-associated TLC1 RNA in both IgG-Sepharose precipitates and
DEAE fractions consistently yielded a shorter fragment of ~70 nt
(Figs. 5, 7B, and 8B). To determine whether the
short fragment was due to an unstable hybrid that can be cleaved
internally by the RNases, we subjected an in vitro
transcript that encompasses residues 1-1301 of the TLC1 RNA to the
same assay. As shown in Fig. 8B, with this RNA, only a
200-nt fragment can be detected. Moreover, the use of a 1-917
transcript, missing the region of TLC1 complementary to the probe,
completely abolished the signal. Taken together, our results indicate
that the RNase protection assay correctly maps the 3' end of the
in vivo and in vitro RNA.

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Fig. 8.
Mapping of the 3' end of
telomerase-associated TLC1 RNA. A, schematic illustration of
the TLC1 gene is presented. The locations of the promoters
and restriction sites utilized in this study, as well as the boundaries
of the probe and in vitro and in vivo
transcripts, are shown. B, TLC1 RNAs derived from DEAE
fractions and in vitro transcription reactions were analyzed
by RNase protection assays. The identities of the RNAs are indicated at
the top. The positions of the major protected fragments are
marked by triangles. The size markers are derived from
MspI digestion of pBR322 DNA. C, TLC1 RNAs
derived from DEAE fractions and in vitro transcription
reactions were polyadenylated and reverse-transcribed with an
oligo(dT)-containing primer. The 3' region of TLC1 cDNA was then
specifically amplified by PCR using a TLC1-internal primer
(corresponding to position 940-965 of the gene) and the same
oligo(dT)-containing primer and analyzed in a polyacrylamide gel. The
size markers are derived from MspI digestion of pBR322 DNA.
D, DNAs from the PCRs were cloned into pBluescript II KS+
and the TLC1-poly(A) junctions determined by sequencing. Two
independent clones derived from the 1-1301 transcript and three
independent clones from the DEAE fractions were sequenced and the
results shown in the figure. The 3' end positions of the RNAs and the
Sm protein-binding site are also indicated.
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|
To further refine the mapping analysis, we sequenced the 3' end of the
in vivo RNA using a protocol developed previously (41). The
RNAs in the fraction were extended by poly(A) polymerase and reverse-transcribed into cDNA using an oligo(dT)-containing primer. The 3' region of TLC1 cDNA was then specifically amplified by PCR,
cloned into a plasmid vector, and sequenced. As a control for the
reliability of the procedure, we analyzed the 3' end of the 1-1301
transcript in parallel. As shown in Fig. 8C, The PCR fragments for the in vivo and in vitro RNA differ
in size by ~130 bp, again consistent with the RNase protection study.
Sequence analysis of independent clones indicate that the in
vivo RNA terminates at either position 1167 or 1168, whereas the
in vitro RNA terminates in a partial XhoI site
after position 1301, precisely as predicted (Fig. 8D). We
conclude that the major 3' end for telomerase-associated TLC1 RNA is
considerably more upstream of the major polyadenylation sites
(~1250), suggesting that during telomerase maturation, the RNA
undergoes both de-adenylation and nucleolytic cleavage.
 |
DISCUSSION |
The Functions of the CP, QFP, and T Motifs--
We have shown in
this study that three of the four conserved N-terminal motifs of yeast
TERT are required for efficient telomerase ribonucleoprotein assembly
but not for enzyme function. Evidence in favor of this proposition
includes the following: 1) the simultaneous loss of telomerase
activity, TERT protein, and TERT-associated RNA in the mutant strains;
2) the inability of the mutant proteins to act in a dominant-negative
fashion when overexpressed in the presence of wild type protein; 3) the
failure of the overexpressed mutant proteins to associate with
telomerase RNA efficiently; and 4) the close quantitative correlation
between telomerase activity and telomerase-associated RNA in the mutant
and overexpression strains. Friedman and Cech (52) have shown earlier
that two 10-amino acid substitutions in region III of yeast TERT
(corresponding to the QFP motif) impair association with TLC1 RNA. Our
results are in agreement and highlight the importance of conserved TERT residues in mediating telomerase assembly.
The mechanistic basis for the observed assembly defect is not clear
from the present analysis; telomerase biogenesis entails multiple
steps, several of which can potentially be disrupted by the mutations.
Based on earlier analysis from several systems, two plausible
hypotheses may be considered. First, direct physical interaction
between yeast TERT and TLC1 RNA may be impaired by the mutations.
Consistent with this idea, the CP, QFP, and T motifs of
Tetrahymena and human TERT have been demonstrated previously (33, 53-55) to be required for binding telomerase RNA in
vitro. Second, relevant intracellular localization of yeast TERT
may be impaired by the mutations, leading to reduced assembly.
Consistent with this latter possibility, residues in the CP and T
motifs of human TERT are believed to mediate nucleolar localization, and nucleolar localization has been proposed to be an important step in telomerase biogenesis (55-57). Further studies will be necessary to distinguish between these (non-mutually exclusive) hypotheses.
The phenotypic consequences of mutations in the CP, QFP, and T motifs
contrast sharply with those of the GQ motif. For instance, some of the
mutations in the GQ region adversely affect telomere maintenance
without affecting telomerase activity (32, 52, 58). In addition,
alteration of conserved residues in the GQ motif can result in mutants
that act in a dominant-negative fashion when overexpressed in the
presence of wild type protein, indicative of some defect in enzyme
function rather than assembly (32). Finally, in complementation
experiments in vitro, telomerase activity can be
reconstituted by mixing an active-site mutant of human TERT with one
that bears mutations in the GQ motif (but not in the other three
motifs) (59). Taken together, these results strongly imply a separate
function(s) for the most N-terminal GQ motif, which apparently resides
in a distinct physical domain (32).
Evolutionary Conservation of the TERT N-terminal Motifs--
The
evolutionary conservation of TERT N-terminal motifs is remarkable and
is consistent with crucial function(s) for these motifs in telomerase
assembly and/or catalysis. As described earlier, an interesting feature
of these motifs is the preponderance of hydrophobic/aromatic residues
at conserved positions. If the CP, QFP, and T motifs are indeed
involved in direct RNA-protein binding, as suggested earlier, then it
is tempting to speculate that this binding may occur primarily through
base-stacking interactions. It is also tempting to speculate that
common features of the telomerase RNAs may be recognized by these
motifs. Conversely, the apparent lack of these motifs in C. elegans TERT suggests that the nature of RNA-protein interaction
may be quite distinct for this telomerase.
The Processing of Telomerase RNA--
The synthesis and maturation
of telomerase RNA appears to be a poorly conserved process. For
example, in ciliates, telomerase RNA is evidently transcribed by RNA
polymerase III, whereas in yeast and mammals, the same function is
mediated by RNA polymerase II (41, 60-62). Human telomerase RNA
appears to be processed by the same factors that process small
nucleolar RNAs with the box H/ACA motif (62, 63). In contrast, the
Saccharomyces TLC1 RNA is bound by Sm proteins and requires
the same proteins for accumulation (64). Understanding the precise
pathways of RNA maturation is clearly an important aspect of
understanding telomerase biogenesis. An earlier study of TLC1 RNA
processing indicates that this RNA is transcribed by RNA polymerase II
and initially polyadenylated (41). The majority of RNA then becomes
de-adenylated, and this appears to be the predominant form that is
present in telomerase RNP. With the exception of the poly(A) tail, the
two RNA populations are believed to have the same 3' ends, clustering around position 1250. However, our current study suggests that the
majority of telomerase-associated TLC1 RNA has a more upstream 3' end,
around position 1167. TLC1 RNA may therefore be subjected to additional
cleavage events duration maturation and RNP assembly. Curiously, the
newly discovered 3' end is only a few nucleotides downstream of the Sm
protein-binding site (Fig. 8D), suggesting that the cleavage
activity may be regulated by the Sm protein complex (64). Such a
cleavage step appears to be common to small nuclear RNAs that associate
with Sm proteins, implying a further parallel between the processing of
these RNAs and TLC1 (65).