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Telomerase Rnas of Different Ciliates Have a Common Secondary Structure and a Permuted Template

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Telomerase Rnas of Different Ciliates Have a Common Secondary Structure and a Permuted Template Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Telomerase RNAs of different ciliates have a common secondary structure and a permuted template Joachim Lingner, Laura L. Hendrick, Thomas R. Cech Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215 USA Telomerase is composed of protein and RNA. The RNA serves as a template for telomere DNA synthesis and may also be important for enzyme structure or catalysis. We have used the presence of conserved sequence elements in the promoter and template regions to amplify by PCR the telomerase RNA genes from six different hypotrichous ciliates: Oxytricha nova, Oxytricha trifallax, Stylonychia mytilis, Stylonychia lemnae, Enplotes aedicalatns, and Euplotes eurystomns. RNaseH cleavage of the O. nova RNA in extracts by use of a complementary oligonucleotide leads to loss of telomerase activity, supporting the identification. Primary sequence and biochemical experiments suggest that the templates of Oxytricha and Stylonychia are circularly permuted relative to that of E. aediculatns. On the basis of the pause sites, the former two add G4T4 during a single primer elongation cycle, whereas E. aedicalatus adds G3T4G. The only primary sequence element outside the template that is conserved between these phylogenetically distant telomerase RNAs is the sequence 5'-(C)UGUCA-3', which precedes the template regions by exactly two bases. We propose a common secondary structure model that is based on nucleotide covariations, a model which resembles that proposed previously for tetrahymenine telomerase RNAs. [Key Words: telomerase; telomere; secondary structure; phylogeny; polymerase; ribonucleoprotein] Received April 22, 1994; revised version accepted June 29, 1994. Telomeres, the physical ends of eukaryotic chromo- plotes crassus (Greider and Blackburn 1989; Shippen- somes, are essential for chromosome stability and con- Lentz and Blackburn 1990; Romero and Blackburn 1991); tribute to chromosome organization within the nucleus telomerase activity in Oxytricha nova and mammalian (for review, see Zakian 1989; Blackburn 1991; Gilson et cells also requires an RNA subunit (Zahler and Prescott al. 1993). They typically consist of simple repetitive se- 1988; Morin 1989; Prowse et al. 1993). In Tetrahymena quences rich in G residues in the strand that runs 5' to 3' the telomerase RNA was shown to serve as a template toward the chromosomal end (e.g., T2G 4 in Tetrahym- for telomere DNA synthesis in vivo (Yu et al. 1990). ena, T4G 4 in Oxytricha, T2AG 3 in humans). Telomeric Other roles for this RNA in telomerase structure or di- DNA is of variable length, ranging from tens of base pairs rectly in catalysis remain speculative. in hypotrichous ciliates to a few hundred in yeast, to The biological functions of stable RNAs depend on hundreds of kilobases in mammals (Klobutcher et al. their secondary and tertiary structure. Comparative se- 1981; Shampay et al. 1984; Moyzis et al. 1988; Kipling quence analysis has proven very powerful for the eluci- and Cooke 1990). dation of RNA structures. In this approach homologous Because most DNA polymerases require a template RNA molecules from phylogenetically diverse organ- and an RNA primer, it is expected that telomeres would isms are sequenced and aligned. Base pairs are identified become shorter with each round of DNA replication by compensatory base changes that maintain comple- (Watson 1972). A mechanism for the complete replica- mentarity (for review, see Pace et al. 1989; Woese and tion of telomeres, involving the non-DNA-templated ad- Pace 1993). By use of this approach, Romero and Black- dition of telomeric repeats to the chromosome ends, was burn (1991) compared seven tetrahymenine telomerase first identified in Tetrahymena by Greider and Black- RNA sequences and proposed a common secondary burn (1985). The enzyme that carries out telomere ex- structure. tension is called telomere terminal transferase or telo- We and others are studying telomere structure and merase (for review, see Blackburn 1992, 1993). It is a function in the hypotrichous ciliate Oxytricha nova ribonucleoprotein with an essential RNA moiety (Grei- (Gottschling and Cech 1984; Gottschling and Zakian der and Blackburn 1987). Telomerase RNA genes have 1986; Price and Cech 1987; Hicke et al. 1990; Gray et al. been cloned from various Tetrahymena species, Glau- 1991; Fang et al. 1993). The macronucleus of this organ- coma chattoni (another tetrahymenine ciliate), and Eu- ism contains -24,000 different gene-sized chromosomes 1984 GENES& DEVELOPMENT8:1984-1998 91994 by Cold SpringHarbor Laboratory Press ISSN 0890-9369/94 $5.00 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Telomerase RNA structure, permuted template each present in 1000 copies on average. The telomerase chromosomes encoding telomerase RNA: the telomeric RNA from another hypotrichous ciliate, Euplotes cras- sequences at each chromosomal end, a sequence provid- sus, is longer and, apart from the template region, shows ing the template for telomere synthesis similar to the no primary sequence similarity to the tetrahymenine te- one identified in E. crassus (Shippen-Lentz and Black- lomerase RNAs (Shippen-Lentz and Blackburn 1990). burn 1990), and an 8-bp promoter element (Fig. 2)present Thus, alignment with the tetrahymenine telomerase in all telomerase RNA genes sequenced so far (Romero RNAs could not be made with confidence. This supports and Blackburn 1991). DNA oligonucleotides were de- the large evolutionary distance between hypotrichs and signed to hybridize to these sequence elements. With Tetrahymena, which is comparable to that separating these oligonucleotides as primers and nuclear DNA as the rat from maize or Chlamydomonas (Fig. 1). template, we attempted to amplify telomerase RNA To learn more about the structure of telomerase gene-specific DNA fragments (see Materials and meth- RNAs, we have cloned the telomerase RNA genes from ods for primer sequences and other details). Certain po- Oxytricha nova, Oxytricha trifallax, Stylonychia myti- sitions of the DNA oligonucleotides were made degen- lis, Stylonychia lemnae, Euplotes aediculatus, and Eu- erate to increase the chances of annealing. Amplified plotes eurystomus and have developed a model for their DNA fragments were analyzed by gel electrophoresis secondary structure. This model is compatible with that and sequenced. The sequences of amplified fragments of tetrahymenine telomerase RNAs proposed by Romero served as the basis for the design of new DNA oligonu- and Blackburn (1991), indicating structural conservation cleotides that were used in further PCR amplifications. over large evolutionary distances. Primary sequence and As a representative example, the cloning strategy for biochemical experiments furthermore indicate that the the O. trifallax telomerase RNA gene is described in templates of Oxytricha and Stylonychia telomerases are more detail: The gene was first amplified between pro- permuted with respect to that of E. aediculatus. The moter and telomerase template with the promoter-spe- former two appear to use the sequence 5'-AAAACCCC- cific primer T8T and the template-specific primer T20. 3' as a template for 5'-TTTTGGGG-3' telomeric repeat Subsequent sequence information was used for the de- synthesis, whereas the latter uses the sequence 5'- sign of T25, an oligonucleotide with sense orientation CAAAACCC-3'. This supports the mechanistic model corresponding to sequences close to the template. The 3' of telomerase involving primer annealing, primer elon- half of the gene was then amplified with T25 and Telpr, gation, and translocation (Greider and Blackburn 1989). an oligonucleotide that was designed to anneal at telo- meres of macronuclear DNA. To confirm the obtained Results sequence information, the transcribed region of the telo- merase RNA gene was amplified, subcloned and se- Cloning of teIomerase RNA genes quenced again in separate PCR amplifications with two We used a PCR strategy to clone the telomerase RNA oligonucleotides (T37 and T38) that flanked the gene. genes from six hypotrichous ciliates. Four sequence ele- For O. nova the 3' portion of the gene was amplified ments were expected to be present on the macronuclear first, with an oligonucleotide (T27) that was based on the 0. trifallax telomerase RNA sequence and the telomere- specific primer Telpr. Amplification of the 5' half of the i Rattus norvegicus 0. nova gene from nuclear DNA was unsuccessful. Artemia safina q Zea mays Therefore, nuclear RNA was isolated and reverse tran- Chlamydomonas reinhardtii scribed in the presence of an oligonucleotide (T33) de- Saccharomyces cerevisiae signed to hybridize to telomerase RNA (position 109 to Neurospora crassa 91). The resulting cDNA was dG-tailed at the 3'-end Paramecium tetraurelia with terminal deoxynucleotidyl transferase, PCR-ampli- Tetrahymena hegewischi fled with the telomerase-specific primer (T33) and a dG- Tetrahymena thermophila tail specific primer (T15), and sequenced. In one case (E. trahymena pyriformis eurystomus), an inverse PCR strategy was used to obtain laucoma chattoni the sequence information of the entire telomerase RNA (see Materials and methods). Euplotes aediculatus 0. nova, 0. trifallax, S. lemnae, S. mytilis, and E. Oxldricha nova aediculatus were found to have, excluding telomeric se- tylonychia pustulata quences, 183-,
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