QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 18 March 2003
doi: 10.1242/jcs.00351

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Möllenbeck, M. Articles by Lipps, H. J. Search for Related Content PubMed PubMed Citation Articles by Möllenbeck, M. Articles by Lipps, H. J.

The telomerase-associated protein p43 is involved in anchoring telomerase in the nucleus

Matthias Möllenbeck^*, Jan Postberg^*, Katrin Paeschke, Michael Rossbach, Franziska Jönsson and Hans J. Lipps

Institute of Cell Biology, University Witten/Herdecke, Stockumer Strasse 10, D-58453 Witten, Germany

View larger version (20K): [in a new window]   Fig. 2. Inhibition of p43 expression by RNAi. (a) Construct used for the inhibition of p43. A map of p43 shows the 238 bp construct cloned into p4440. Bacteria were transfected with this construct and the expression of dsRNA was induced as described previously (Timmons and Fire, 1998). Euplotes were fed daily with bacteria. The position of the La motif, the RNA-binding motif (RRM with the submotifs RNP1 and 2) and primers used for PCR amplification are indicated (Aigner et al., 2000). (b) In situ staining of macronuclei with an anti-p43 antibody before (left) and after (right) RNA inhibition. (c) Western blot analysis of cells before (left) and after inhibition (right) of p43 expression. Filters were incubated simultaneously with an anti-p43 antibody and as a control with an anti--tubulin antibody (Sigma). (d) FISH analysis of telomerase RNA subunit after inhibition of p43 before (left) and after (right) electroelution. Control macronuclei (left), macronuclei after inhibition of p43 expression (right). Arrows point to the replication band. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 3. Inhibition of the catalytic subunit of telomerase by RNAi. (a) Construct used for the inhibition of telomerase. A map of the gene encoding the catalytic subunit of telomerase (p123) is shown. The 573 bp construct cloned into p4440 and the primers used are indicated. Motifs CP, T, 1, 2, A, B', C, D and E are shown (Bryan et al., 1998). (b) Before (left) and after (right) inhibition. (c) Distribution of p43 in the macronucleus after inhibition of telomerase expression (left), electroeluted macronuclei after inhibition of telomerase expression stained with an anti-p43 antibody (right). Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 1. Distribution of telomeric sequences, p43 and telomerase in the macronucleus of Euplotes before and after electroelution as described previously (Postberg et al., 2001). Telomeric sequences and telomerase were detected by FISH analysis, p43 by antibody staining. Arrows point to the replication band. (a) Distribution of telomeric sequences, p43 and telomerase in the macronucleus of Euplotes before electroelution. 1, telomeric sequences; 2, p43; 3, telomerase. (b) Distribution of telomeric sequences, p43 and telomerase in the macronucleus of Euplotes after electroelution. 1, telomeric sequences; 2, p43; 3, telomerase. Bar, 10 µm.

View larger version (38K): [in a new window]   Fig. 4. Binding of the p43-telomerase complex to the nuclear matrix. (a) In situ staining of macronuclear halos with DAPI (left) and an anti-p43 antibody (right). (b) Macronuclear halos stained with DAPI (left) and FISH analysis of the telomerase RNA subunit (right). p43 staining after electroelution (c) and after RNAse treatment followed by electroelution (d). Macronuclear halos before (e) and after inhibition of p43 expression by RNAi (f) stained with DAPI (left) and FISH analysis of the telomerase RNA subunit (right). Bar 10 µm.

View larger version (21K): [in a new window]   Fig. 5. Model for the organization of the end-replication machinery in the macronucleus of Euplotes. Telomeric sequences are bound to the nuclear matrix by an interaction of the telomere-binding protein (TEBP) with components of this structure. Similarly, the p43-telomerase complex (p43, TERT) is also bound to this structure. In the course of replication the interaction of telomeres with the nuclear matrix is resolved, and at this stage it may well be that the telomere-binding protein is replaced by a replication-specific binding protein (Carlson et al., 1997). In contrast, the enzymatic machinery stays bound to this structure during replication.