A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains

Abstract

The ribonucleoprotein (RNP) enzyme telomerase is required for replication of eukaryotic chromosomal termini. The RNA moiety of telomerase is essential for enzyme function and provides the template for telomeric DNA synthesis. However, the roles of its nontemplate domains have not been explored. Here we demonstrate that a novel interspecies telomerase RNA swap in vivo creates a functional but aberrant telomerase. Telomerase RNA from the ciliate Glaucoma chattoni was expressed in Tetrahymena thermophila cells. The telomerase RNAs from these two species have almost superimposable secondary structures. The template region base sequence is identical in the two RNAs, but elsewhere their sequences differ by 49%. This hybrid telomerase RNP was enzymatically active but added only short stretches of telomeric repeat tracts in vivo and in vitro. This new enzyme also had a strong, aberrant DNA cleavage activity in vitro. Thus, molecular interactions in the RNP involving nontemplate RNA domains affect specific aspects of telomerase enzyme function, raising the possibility that they may regulate telomerase activity.The G-rich telomeric DNA tracts found at most eukaryotic chromosomal termini are added by a specialized component of the cellular DNA replication machinery, the ribonucleoprotein (RNP) reverse transcriptase telomerase. Telomeric DNA has numerous important functions and properties, including allowing completion of DNA replication, mediating chromosome stability (1), and affecting mitotic chromosome separation (2). Therefore, telomere synthesis and maintenance are crucial aspects of stable genomic inheritance in eukaryotes. The RNA moiety of the telomerase RNP is essential for enzymatic function and contains an internal sequence that templates telomeric DNA polymerization (3). Studies of template point mutations also have revealed other critical roles for specific template residues in enzyme action (4, 5). However, little is known about the function of other portions of telomerase RNA.Telomerase RNAs in ciliated protozoa (3, 6, 7), yeasts (8, 9), and mammals (10, 11) have diverged rapidly in primary sequence. Phylogenetic (12-14) and RNA conformational analyses (15, 16) of the small (Ϸ150-200 nt) ciliate telomerase RNAs have shown that their secondary structures are highly conserved. Comparison of 13 telomerase RNAs from one group of ciliates has shown a strikingly bipartite sequence arrangement; they all contain a 17-nt stretch of identical sequence centered on the template domain, and the primary sequence of the rest of the RNA differs by up to 76% yet preserves similar structural domains (12,14). To investigate directly how this nontemplate portion of telomerase RNA affects enzymatic function, we tested whether the Ϸ160-nt telomerase RNAs in the ciliates Tetrahymena thermophila and Glaucoma chattoni were functionally interchangeable in vivo. These two telomerase RNAs share an identical 23-base sequence in and around the template domain. In contrast, the rest of the RNA sequence, which has an almost identic...

Flexible Positioning of the Telomerase-Associated Nuclease Leads to Preferential Elimination of Nontelomeric DNA

“…Telomerase particles reconstituted with wild-type RNA cleave primers only at the previously defined site (1). In contrast, telomerase complexes containing Tetrahymena proteins reconstituted with Glaucoma chattoni telomerase RNA exhibit aberrant nuclease activity in vitro, cleaving primers at other positions (5). The chimeric telomerase also displays reduced processivity compared to the wild-type telomerase (5).…”

“…In contrast, telomerase complexes containing Tetrahymena proteins reconstituted with Glaucoma chattoni telomerase RNA exhibit aberrant nuclease activity in vitro, cleaving primers at other positions (5). The chimeric telomerase also displays reduced processivity compared to the wild-type telomerase (5). Hence, the cleavage and processive elongation may be mechanistically linked as is seen with RNA polymerase II (19,34,37).…”

“…Telomerase nuclease activity can remove both telomeric and nontelomeric residues from a primer 3Ј terminus (1,5,6,28,33). Previous studies indicated that cleavage removes nucleotides that extend across the 5Ј boundary of the RNA template, whether they can form Watson-Crick base pairs with the telomerase RNA or not ( Fig.…”

“…These events are developmentally programmed and temporally coupled during the formation of the new macronuclear genome following conjugation (reviewed in reference 8). It has also been postulated that cleavage serves a proofreading function (6,28,33) and may be mechanistically linked to processivity (5).…”

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

“…With an RNP gel shift assay, the highly conserved pseudoknot in the telomerase RNA is important for tTERT binding but is not required for association with the Tetrahymena telomerase-associated proteins p80 or p95 Greider, 1994, 1995;Gilley and Blackburn, 1999;Licht and Collins, 1999). In the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, certain mutations outside the telomerase RNA template domain are critical for telomerase RNP assembly, polymerization, and telomere length maintenance in vivo (McEachern and Blackburn, 1995;Bhattacharyya and Blackburn, 1997;Prescott and Blackburn, 1997b;Roy et al, 1998). These studies illustrate a critical role for the telomerase RNA distinct from its templating function in telomerase catalysis in humans, ciliates, and yeast.…”