Polymerization Defects within Human Telomerase Are Distinct from Telomerase RNA and TEP1 Binding

Abstract

The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs. INTRODUCTIONTelomeres are the specialized nucleoprotein complexes at the physical ends of eukaryotic chromosomes (Greider and Blackburn, 1996). Telomerase is a ribonucleoprotein (RNP) enzyme that uses an internal RNA template to specifically direct telomere synthesis (Greider and Blackburn, 1985, 1987). Telomerase plays an essential role in the dynamic process of telomere length regulation in vivo by restoring telomeric sequences that are lost during semiconservative DNA replication (Watson, 1972;Olovnikov, 1973). Telomerase activity has been purified from a number of different organisms, including mammals (Greider, 1996). The telomerase RNA component (TER, telomerase RNA) has been cloned from many different species (Greider, 1996), and the secondary structure of the ciliate and mammalian telomerase RNAs is highly conserved (Romero and Blackburn, 1991;Lingner et al., 1994;McCormick-Graham and Romero, 1995;Chen et al., 2000).The first mammalian telomerase proteins (TEPs) were identified based on homology with previously cloned telomerase components from ciliates and yeast. TEP1 is a Tetrahymena p80 homolog that binds TER and is associated with telomerase activity in cell extracts (Collins et al., 1995;Harrington et al., 1997a;Nakayama et al., 1997). The mammalian telomerase reverse transcriptase (TERT) shares amino acid sequence similarity with the catalytic telomerase subunit previously identified in ciliates and yeast (Harrington et al., 1997b;Kilian et al., 1997;Lingner et al., 1997;Meyerson et al., 1997;Nakamura et al., 1997). Human TERT is a limiting component for telomerase activity: the hTERT mRNA is often up-regulated in cells containi...

Stabilization of Telomere Length and Karyotypic Stability Are Directly Correlated with the Level of hTERT Gene Expression in Primary Fibroblasts

“…4A, lane 9). Although initially surprising to us, this observation is consistent with recent reports using a different hTERT antibody in Western blotting experiments, which, again, failed to detect hTERT protein in 293 cells, despite high levels of telomerase catalytic activity (23,24). Protein expression was also investigated by immunocytochemistry.…”

Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

“…In separate titration experiments, we found no effect of increasing levels of mPif1 on human telomerase or murine telomerase, by TRAP or a non-PCR-based elongation assay (data not shown). To further test the potential effect of mPif1 on telomerase activity, we immunoprecipitated wild-type Pif1 and a predicted helicase-inactive variant of Pif1 with both wild-type full-length hTERT and a truncation of hTERT that we showed previously was defective in processive elongation (⌬200) (5,6). Because 100% of the telomerase in the anti-HA immunoprecipitation was associated with HA-Pif1, these results allow us to conclude that neither wild-type nor mutant mPif1 affects telomerase activity when specifically bound to TERT (Fig.…”

“…The reconstitution of murine or human telomerase was performed as described previously (6, 7). The reaction mixtures were then immunoprecipitated with 25 l of a 50% (vol/vol) slurry of either anti-HA (3F10) agarose beads (Roche), anti-Flag beads (Sigma Aldrich Co.), or protein A-Sepharose-CL4B (Sigma Aldrich Co.) coupled with polyclonal antibody, washed, and resolved as described previously (6,7). Western blots were probed with anti-HA (3F10), anti-mPif1, or anti-TERT antibody and detected by chemiluminescence (Amersham Pharmacia).…”

Mechanisms of cell immortalization mediated by EB viral activation of telomerase in nasopharyngeal carcinoma

“…Being the catalyst interacting with both hTERC and telomeric DNA, hTERT contains four major regions: an N-terminal regulatory (R) domain, RNA-binding (RB) domain, reverse transcription (RT) domain and C-terminal dimerization domain [45]. Several motifs in the R and RB domains of hTERT are conserved in different species, important for various molecular interactions [41,[46][47][48]. As the rate-limiting component of telomerase, hTERT induces immortalization of a number of cell types in culture [49] (reviewed in [50]).…”