Institute of Cell Biology, University Witten/Herdecke, Stockumer Strasse 10, D-58453 Witten, Germany
Author for correspondence (e-mail:
lipps{at}uni-wh.de)
Accepted 7 January 2003
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Summary |
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Further analysis revealed that the p43-telomerase complex is bound to the nuclear matrix in vivo and that after inhibition of p43 expression, telomerase is released from this structure, strongly suggesting that p43 is involved in anchoring of telomerase in the nucleus. This is the first in vivo demonstration of the biological function of this telomerase-associated component involved in telomere replication and allows us to propose a model for the organization of the end-replication machinery in the eukaryotic cell.
Key words: Ciliates, La autoantigen, Nuclear matrix, Replication factory, Telomerase, RNAi
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Introduction |
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We have recently shown that telomeric sequences in the spirotrichous
ciliate Stylonychia lemnae adopt the anti-parallel G-quartet
structure in vivo (Schaffitzel et al.,
2001) and are bound in this structure to the nuclear matrix by a
specific interaction of the TeBP with components of this subnuclear structure.
In the course of replication both the G-quartet structure as well as the
interaction of the telomere-binding protein with the nuclear matrix is
resolved, making telomeres accessible to telomerase action
(Jonsson et al., 2002
;
Postberg et al., 2001
). In the
related species Euplotes aediculatus, telomerase is associated with
the protein p43 (Aigner et al.,
2000
). This protein copurifies with active telomerase and appears
to be stoichiometric with both the RNA and the catalytic subunit of the
telomerase complex. Recombinant p43 binds telomerase RNA in vitro. It shares
homology with the La autoantigen family
(Aigner et al., 2000
), which
has been shown to be involved in sequestering RNA in the nucleus
(Maraia, 2001
) and to bind to
internal sequences and/or structures (Chang
et al., 1994
; Grimm et al.,
1997
) in vitro. It therefore has been suggested that p43 functions
in the assembly and/or nuclear retention of telomerase
(Aigner et al., 2000
).
In this report we analyze the organization of the p43-telomerase complex in the macronucleus of Euplotes and show that both components are bound in vivo to a subnuclear structure, the nuclear matrix or chromosome scaffold. But in contrast to telomeric sequences this complex is not released from this structure during replication. Using the powerful tool of RNAi technology we now can show that one of the major functions of the telomerase-associated protein p43 is to contribute to nuclear retention of telomerase and anchorage of this enzyme complex in the replication factory of the ciliate macronucleus. Furthermore, we made an attempt to analyze the molecular interaction involved in this retention mechanism.
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Materials and Methods |
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Isolation of nuclei
For isolation of macronuclei, cells were lysed with 2x lysis buffer
(1 mg/ml spermidine phosphate, 0.1% Triton X-100) and centrifuged at 500
g for 7 minutes.
Halo preparation
Macronucleus halos were isolated using the lithium diiodosalicylate (LIS)
technique described for mammalian cells
(Dijkwel and Hamlin, 1988),
although the light microscopical appearance is very different from mammalian
nucleus halo preparations owing to the small size of macronuclear DNA
(Jonsson et al., 2002
).
Furthermore because of mechanical forces during preparation the elongated
macronuclei become fragmented in most of the cases.
Inhibition of gene expression by RNAi
Constructs used for the inhibition of p43 and telomerase were obtained by
polymerase chain reaction (PCR) using the following primers
(Fig. 2a and
Fig. 3a):
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Inhibition of gene expression was achieved by feeding Euplotes
with E. coli cells HT115 (F-, mcrA, mcrB,
IN(rrnD-rrnE)1, lambda-, rnc14::Tn10(DE3 lysogen:lacUV5 promoter-T7
polymerase) expressing the corresponding dsRNAs using vectors as
described previously (Timmons and Fire,
1998). Usually a first effect could be observed after about three
days of feeding. As controls, Euplotes were fed only with bacteria or
bacteria containing the vector without insert.
In situ localization, electroelution and western blot analysis
In situ localization of telomeres, p43 and the telomerase, electroelution
experiments and western blot analyses were done as previously described for
Stylonychia (Jonsson et al.,
2002; Postberg et al.,
2001
). The probe used for FISH analysis of the RNA telomerase
subunit was the digoxigenin-labeled oligonucleotide 5'-(DIG-C12)
rm(GCUUGACAGAUUCUACA)dG-3'.
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Results and Discussion |
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Subsequently, expression of the catalytic subunit of telomerase (p123)
(Lingner et al., 1997) was
inhibited using RNAi directed against a sequence encoding 191 amino acids
located at the C-terminus of telomerase
(Fig. 3a), and the degree of
inhibition was indirectly demonstrated by FISH analysis using a probe directed
against a non-template region of the RNA subunit
(Fig. 3b). Upon inhibition of
telomerase, p43 distribution in the macronucleus is strongly disturbed and no
longer organized in foci-like structures and can be completely electroeluted
from the nucleus (Fig. 3c). In
common with p43 inhibition, telomerase inhibition leads to loss of DNA from
the nucleus, fragmentation of nuclei and eventually to the death of the cells.
Although the overall morphology of macronuclei is still visible after
inhibition of p43 or telomerase followed by electroelution, owing to the loss
of a high amount of DNA no adequate DAPI staining can be obtained and,
unfortunately, in these organisms no antibodies against matrix proteins are
available. At this point it could be speculated that upon binding of
telomerase to p43 this molecule undergoes a structural modification, and it is
only after this modification that p43 can bind to the nuclear matrix. This
would ensure that it is only the p43 in the p43-telomerase complex that can
bind, although these binding sites cannot be occupied by free p43. This view
is supported by the observation that p43 is stoichiometric with telomerase in
the nucleus, that it always seems to be complexed with p43 in the macronucleus
(Aigner et al., 2000
) and in
situ localization experiments never revealed a p43 signal without an
overlapping signal for telomerase.
To distinguish between all the possibilities discussed above and to analyze the nature of interactions involved in telomerase retention, we first prepared macronuclear halos, which are relaxed lengths of looped DNA with attachment to the nuclear matrix. However, because of the small size of macronuclear DNA the light microscopical appearance of Euplotes macronuclear halos is very different from mammalian nuclear halo preparations. In this structure we tested by in situ antibody staining and FISH analysis whether the p43-telomerase complex copurifies with this structure. As shown in Fig. 4a,b, both components are bound to the nuclear matrix strongly suggesting that nuclear retention is due to an interaction of this complex with components of this structure. Upon inhibition of p43 by RNAi, telomerase is no longer retained in the macronucleus. Similarly, upon inhibition of the telomerase catalytic subunit p43 distribution in the macronucleus is also disturbed and can be electroeluted (Fig. 3a,b). To test the hypothesis that binding of the telomerase RNA subunit to p43 is required for binding of p43 to the nuclear matrix we treated macronuclei with RNAse and studied the behaviour of p43 after this treatment. As shown in Fig. 4c,d under these conditions p43 is efficiently electroeluted from the nucleus. Although the overall morphology of macronuclei after RNAse treatment did not change at the light microscopical level, some as yet unknown modifications of nuclear structure cannot be excluded. However, these experiments strongly indicate that it is indeed the p43-RNA complex that binds to the nuclear matrix. To further strengthen this assumption we inhibited expression of p43 by RNAi and tested in halo preparations whether telomerase is still attached to the nuclear matrix. Fig. 4e,f shows that this is no longer the case, demonstrating that telomerase itself does not bind to the nuclear matrix, but that p43 acts as an adaptor protein, which only after binding to the RNA subunit of the telomerase, adopts a modification in which it can bind to this subnuclear structure.
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Taking all these data together, it is evident that only the p43-telomerase
complex binds to a subnuclear structure, and any disturbance of one of these
components leads to the release of the other. Furthermore, our data allow us
to propose a model for the organization of the end-replication machinery
during the replication process (Fig.
5). In the course of replication, the G-quartet structure of
telomeric sequences as well as the interaction of the telomere-binding protein
with the nuclear matrix is resolved, releasing the DNA
(Jonsson et al., 2002;
Postberg et al., 2001
). It
might well be that the telomere-binding protein is replaced by a
replication-specific binding protein, as suggested earlier
(Carlson et al., 1997
). By
contrast, in the replication band the enzymatic machinery stays bound to this
subnuclear structure, a phenomenon that has been suggested to be true for
other enzymes involved in replication and transcription in mammalian
replication and transcription factories
(Cook, 1999
).
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Acknowledgments |
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Footnotes |
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