Functional Multimerization of the Human Telomerase Reverse Transcriptase

The catalytic subunit of telomerase, the telomerase reverse transcriptase (TERT), possesses the hallmark amino acid motifs of a reverse transcriptase (RT) (23, 29, 40, 47). However, unlike viral RTs, telomerase is a unique eukaryotic RT that carries an intrinsic RNA template essential for the de novo addition of telomere sequences (reviewed in reference 19). Proteins associated with telomerase activity include TEP1 (22, 48), hsp90/p23 (18, 25), dyskerin (42, 43), L22 (32), and hStau (32) in mammals; the Sm proteins (54) as well as Est1p and Est3p, (26, 56) in Saccharomyces cerevisiae; and p80, p95, and p43 in ciliates (1, 11, 21, 35). TEP1 is not essential for telomerase activity in vitro or in vivo (5, 37). A subset of these associated factors are known to serve distinct roles in telomerase assembly and telomere length maintenance (15, 16, 27, 34, 41, 43, 51, 54). In S. cerevisiae, different telomerase RNAs can functionally cooperate to form an active telomerase complex in vivo. Prescott and Blackburn demonstrated the presence of at least two primer recognition-elongation sites within S. cerevisiae telomerase (50). In addition, they showed that a mutant telomerase RNA incapable of telomere elongation could nonetheless support elongation in a diploid strain containing one mutant and one wild-type telomerase RNA (50). These results provided the first evidence that telomerase could form an active multimer in vivo that might contain, at minimum, two active sites (50). A recombinant reconstitution assay for human telomerase showed that two separately inactive, nonoverlapping fragments of human telomerase RNA could reconstitute telomerase activity in vitro (59). While consistent with a model of telomerase RNA multimerization, these results are also consistent with reconstitution of a single active telomerase RNA from two inactive telomerase RNA fragments. In vitro, the minimal requirements for telomerase activity appear to comprise the telomerase RNA and human TERT (hTERT) (3, 6, 9, 60). Previously, we found that the first 300 amino acid residues (aa) of hTERT were dispensable for telomerase activity in vitro and in vivo (5) (summarized in Fig. ​Fig.1A).1A). However, the telomerase activities associated with N-terminal truncations of hTERT were severely reduced in rabbit reticulocyte lysates (RRLs) relative to the activities achieved when the same hTERT truncation proteins were introduced into telomerase-positive 293T cells (5). Furthermore, deletion of the C-terminal 204 aa of hTERT did not affect telomerase activity in 293T cells, whereas all but the C-terminal 20 aa are absolutely required for telomerase activity in RRL (4, 5) (summarized in Fig. ​Fig.1A).1A). In this study, we set out to determine whether the observed discrepancies in activity between truncated hTERT proteins in RRL and 293T cells might be explained by the multimerization of specific hTERT fragments with endogenous hTERT. FIG. 1 Physical and functional interactions of full-length and truncated hTERT proteins in vitro. (A) A schematic diagram of full-length hTERT, including a summary of the minimal fragments of hTERT that are sufficient for telomerase activity in RRL and 293T ...