Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

Telomeres protect authentic chromosome termini from aberrant resection or rearrangement. In most eukaryotes, a stable telomere is composed of a tandem array of simple-sequence repeats assembled into specialized chromatin (37). The overall length of the telomeric simple-sequence repeat tract is maintained in a dynamic balance of sequence loss and addition. To compensate for the inevitable loss from incomplete replication by DNA-dependent DNA polymerases, telomeres recruit the telomerase ribonucleoprotein (RNP) reverse transcriptase in coordination with other DNA replication factors (18). Telomerase copies a defined sequence within its integral RNA subunit (TER) to extend chromosome 3′ ends. Sequence substitutions within the TER template can rapidly impose defects in cell division and viability due to the synthesis of altered repeats (6). During a catalytic cycle of repeat synthesis, the telomerase active site must proceed through a remarkable number of distinct states of template positioning and pairing (Fig. ​(Fig.1A).1A). Substrates unable to base-pair with the template are extended in alignment with the template 3′ end at its “default” position, defined as the position of template in the absence of a template-primer hybrid (41). For Tetrahymena thermophila TER, default template positioning places C49 in the active site (Fig. ​(Fig.1A,1A, top). As deoxynucleoside triphosphates (dNTPs) are added, a template-product hybrid forms, and successively more 5′ positions of unpaired template must thread into the active site (Fig. ​(Fig.1A,1A, middle). Product synthesis halts at the template 5′ boundary, corresponding to position C43 of T. thermophila TER (Fig. ​(Fig.1A,1A, bottom). Upon eventual dissociation of the template-product hybrid, the liberated template repositions to recover its default placement. Product either dissociates from the enzyme or repositions at the template start for processive repeat addition. FIG. 1. Structure and expression of TER. (A) Selected stages of the telomerase catalytic cycle. Internal template boundaries are influenced by TERT-TER interactions that restrict template motion, illustrated here as small ovals. The default positioning of template ... Two telomerase holoenzyme subunits have direct roles in catalysis: TER and the telomerase reverse transcriptase protein (TERT). TER domain structure has diverged between ciliates, vertebrates, and yeasts, with the possible exception of a template-adjacent pseudoknot (3, 8). TERT harbors a set of active site motifs shared by all reverse transcriptases and an N-terminal extension conserved only among TERTs (21). The TERT N-terminal extension contains motifs necessary for high-affinity interaction with TER and for recruitment to telomere substrates. Recent studies have exploited rabbit reticulocyte lysate (RRL) for the reconstitution of a minimal Tetrahymena or human telomerase RNP from TERT and TER alone. Using this in vitro system, motifs that influence specific features of catalytic activity have been defined for Tetrahymena TER (23-25, 28, 30, 32, 38) and for human TER as well (7, 9). Telomerase holoenzyme assembly in vivo could influence TER motif functions previously defined using the minimal RNP assembled in vitro. The holoenzyme contains many subunits in addition to TERT and TER, including an RNA binding protein(s) that alters TER folding (42, 35). Nontemplate substitutions within yeast TERs reconstituted in vivo can alter telomerase catalytic properties, with associated defects in telomere length maintenance (reference 40 and references therein). Here, we investigate nontemplate TER motif functions in the Tetrahymena telomerase holoenzyme reconstituted in vivo. We examined TER motif functions in the holoenzyme catalytic cycle using direct primer elongation activity assays, combined with an RNA-based RNP affinity purification strategy to enrich telomerase holoenzyme from cell extracts. We also determined nontemplate TER motif requirements for telomerase function on its telomere substrates. We find that TER sequence substitutions affect the equilibrium set point for telomere length maintenance in vivo and induce specific changes in the catalytic activity of telomerase holoenzyme in vitro. Our findings demonstrate that nontemplate motifs of Tetrahymena TER have specific roles in telomerase holoenzyme function.