Human telomerase: from structure and function to telomere recruitment

The telomerase holoenzyme consists of an RNA moiety, which is associated with several protein subunits. The catalytic protein subunit is referred to as TERT (TElomerase Reverse Transcriptase). It is structurally and functionally related to retroviral reverse transcriptases (Fig. 1). TERT synthesizes telomeric repeats by reverse transcribing in an iterative fashion a specific sequence of the telomerase RNA onto chromosome 3' ends. In human, telomerase is associated with additional proteins including dyskerin and Gar1, both of which are also associated with small nucleolar RNPs; and the heat shock proteins p23 and Hsp90, which may promote assembly of telomerase. However, other components of human telomerase are likely to exist and remain to be discovered. Most prominently, a human homologue of the yeast Est1 protein, which is thought to be responsible for guiding telomerase to chromosome ends, remains to be discovered. From gel filtration analyses we estimate that the telomerase holoenzyme has a molecular weight of 3,000,000 Da.

We have established a robust system to reconstitute active human telomerase from recombinant telomerase reverse transcriptase (TERT) expressed in insect cells and in vitro transcribed telomerase RNA (Wenz et al., 2001). We have found that the native molecular weight of the recombinant enzyme corresponds to a dimer, and that every telomerase complex contains two telomerase RNA molecules. Significantly, a telomerase-heterodimer containing one wild type and one mutant telomerase RNA template was virtually inactive by comparison to the wild type homodimer. This indicates that the telomerase RNA templates in the active enzyme are interdependent and functionally cooperate with each other. We have proposed models that may explain the biological and enzymatic roles of telomerase dimerization. In the first model, which we refer as a template-switching model, the extension of the telomere 3' end during processive synthesis is accompanied by switching of the template after the addition of a telomeric repeat. Thus, translocation of the extended telomere 3' end would involve a concomitant switch of the active site in the dimer. In the second model, which we refer to as a parallel extension model, two active sites in the telomerase-complex extend two separate telomere 3' ends in a cooperative fashion. This model could provide a means for the parallel extension of sister chromatid 3' ends. In the third model, which is referred to as anchor-site model, one subunit uses its template primarily for binding of the substrate while the second template functions in catalysis and reverse transcription. This could allow translocation of the telomere 3' end in the active site while preventing its dissociation from telomerase during translocating from one end of the template to the other.

Figure 1: Model of telomerase as an RNA-reverse transcriptase complex.
The reverse transcriptase domain (green) is based on HIV1-RT. The RNA subunit (purple) is shown schematically in its secondary structure representation. The telomeric DNA substrate is in red. Modified from Lingner, J., Hughes, T.R., Shevchenko, A., Mann, M., Lundblad, V., and Cech, T.R. (1997). Science 276, 561-567.

Future work concentrates on the following major topics:

(1) We are performing experiments to elucidate the function of telomerase dimerization and to characterize the interaction. This will lead to a better understanding of the telomerase reaction mechanism.

(2) We are using the recombinant active telomerase (hTERT-hTER) complexes to study the mechanism of action of previously identified and novel telomerase- and telomere-associated proteins in vitro. This should uncover their putative roles in regulating telomerase activity and the access to the telomere.

(3) We will assess the function of putative telomere replication factors in vivo by using RNA interference technology.

(4) Recombinant telomerase provides opportunities to study the mechanism of action of telomerase inhibitors. We have been characterizing in collaboration with Boehringer-Ingelheim a small molecule that inhibits telomerase activity in vitro and in vivo (Damm et al. 2001; Pascolo et al. 2002).

Regulation of hTERT expression

In most tumors, hTERT is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. In collaboration with the groups of Markus Nabholz (ISREC), Robert Newbold (Brunel University) and others we have studied the regulation of this gene. These studies are described in recent publications (Wu et al. 1999; Szutorisz et al. 2001; Ducrest et al., 2001; 2002).

Biogenesis of yeast telomerase

In S. cerevisiae, the telomerase holoenzyme contains the telomerase reverse transcriptase subunit Est2p, the telomerase RNA moiety TLC1, the telomerase associated proteins Est1p and Est3p and Sm proteins. In collaboration with Susan Gasser (University of Geneva) we assessed telomerase assembly by determining the localization of telomerase components (Teixeira et al., 2002). We found that Est1p, Est2p and TLC1 can migrate independently of each other to the nucleus. With limiting amounts of TLC1, overexpressed Est1p and Est2p accumulated in the nucleolus whereas enzymatically active Est2p-TLC1 complexes were distributed over the entire nucleus. The distribution to the nucleoplasm depended on the specific interaction between Est2p and TLC1 but was independent of Est1p and Est3p. These results suggest a role of the nucleolus in telomerase biogenesis. We also performed experiments that support a transient cytoplasmic localization of TLC1 RNA. Thus, distinct cellular compartments are involved in yeast telomerase biogenesis.

Telomere sequence analysis in S. cerevisiae: assessing telomere extension in vivo

We developed a PCR-method that allows efficient telomere length and sequence analysis (Förstemann et al. 2000). Because of the degeneracy of yeast telomeres, this method also allowed distinction of two zones within a telomere, a centromere-proximal zone that is replicated by semi-conservative DNA replication, and a centromere-distal zone in which telomere shortening and telomerase-mediated extension occurs. Since the distal domain is much larger than the number of nucleotides lost per generation in the absence of telomerase, this finding suggested that telomerase does not extend each telomere in every cell cycle. Based on a detailed analysis of the telomere sequences specified by wild type and mutant RNA templates in vivo, we have also elucidated the mechanisms by which the single telomerase RNA template from
S. cerevisiae specifies synthesis of multiple telomere repeats (Förstemann and Lingner, 2001). In genetic screens we identified template sequence mutants that give rise to temperature-sensitive growth. In these cells, either the mutant telomeres become uncapped when the cells are shifted to the restrictive temperature or the telomerase enzyme becomes inactive at the elevated temperature.

Future work focuses on:

(1) the identification of suppressors of the temperature sensitive telomerase mutants. We expect to identify components involved in telomere replication and/or telomere capping.

(2) We are studying telomerase recruitment and extension efficiency in vivo in wild type cells and in mutant backgrounds by exploiting the telomere-PCR method.

Collaborations

These projects are carried out in part as a collaboration with the groups of Markus Nabholz, ISREC, Philipp Bucher, Swiss Institute of Bioinformatics, Julie Cooper, CRUK, Robert Newbold, Brunel University, Klaus Damm, Boehringer-Ingelheim, Daniela Rhodes, MRC, Susan Gasser, University of Geneva, and Thomas R. Cech, University of Colorado

PhD Theses (University of Lausanne)

Klaus Förstemann (2002).
Molecular mechanisms of telomere elongation by budding yeast telomerase.

Anne-Lyse Ducrest (2002).
Regulation of the human telomerase reverse transcriptase gene.