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• W2009099360 abstract "Telomerase is the cellular RNA-dependent DNA polymerase (i.e. reverse transcriptase) that uses an integral RNA template to synthesize telomeric DNA repeats at the ends of linear chromosomes. Human telomerase RNA (hTERC) is thought to function as a dimeric complex consisting of two RNAs that interact with each other physically as well as genetically. We show here for the first time that the yeast Saccharomyces cerevisiae telomerase RNA TLC1 likewise forms dimers in vitro. TLC1 dimerization depends on a unique 6-base self-complementary sequence, which closely mimics palindromic sequences that mediate functional dimerization of HIV-1 and other retroviral genomes. We found that dissimilar but comparably located TLC1 palindromes from other sensu stricto yeasts can functionally substitute for that of S. cerevisiae. Yeast cells expressing dimerization-defective TLC1 alleles have shorter telomeres than those with wild-type TLC1. This study, therefore, highlights dimerization as a functionally conserved feature of the RNA templates utilized by reverse transcriptases of both viral and cellular origins. Telomerase is the cellular RNA-dependent DNA polymerase (i.e. reverse transcriptase) that uses an integral RNA template to synthesize telomeric DNA repeats at the ends of linear chromosomes. Human telomerase RNA (hTERC) is thought to function as a dimeric complex consisting of two RNAs that interact with each other physically as well as genetically. We show here for the first time that the yeast Saccharomyces cerevisiae telomerase RNA TLC1 likewise forms dimers in vitro. TLC1 dimerization depends on a unique 6-base self-complementary sequence, which closely mimics palindromic sequences that mediate functional dimerization of HIV-1 and other retroviral genomes. We found that dissimilar but comparably located TLC1 palindromes from other sensu stricto yeasts can functionally substitute for that of S. cerevisiae. Yeast cells expressing dimerization-defective TLC1 alleles have shorter telomeres than those with wild-type TLC1. This study, therefore, highlights dimerization as a functionally conserved feature of the RNA templates utilized by reverse transcriptases of both viral and cellular origins. Telomerase is the cellular reverse transcriptase (RT) 2The abbreviations used are: RT, reverse transcriptase; nt, nucleotide(s); EMSA, electrophoretic mobility shift assay; HIV, human immunodeficiency virus. 2The abbreviations used are: RT, reverse transcriptase; nt, nucleotide(s); EMSA, electrophoretic mobility shift assay; HIV, human immunodeficiency virus. that is responsible for adding telomeric DNA repeats onto the ends of linear chromosomes (1Blackburn E.H. Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1750) Google Scholar). Found in all eukaryotes, it is a multimolecular complex of which the minimal components are a catalytic RT protein (TERT) and an integral RNA template. TERT proteins from diverse mammalian, protozoan, and yeast species appear to be related to one another evolutionarily as well as to RTs from retroviruses and cellular retroelements (2Lingner J. Hughes T.R. Shevchenko A. Mann M. Lundblad V. Cech T.R. Science. 1997; 276: 561-567Crossref PubMed Scopus (1032) Google Scholar, 3Peng Y. Mian I.S. Lue N.F. Mol. Cell. 2001; 7: 1201-1211Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 4Feng Y.X. Moore S.P. Garfinkel D.J. Rein A. J. Virol. 2000; 74: 10819-10821Crossref PubMed Scopus (39) Google Scholar). Telomerase RNAs, by contrast, differ widely in length and primary sequence, and their phylogenetic relationships to one another and to retroviral genomes, if any, are unclear. Certain properties that are shared among groups of retroviral or telomerase RNAs may indicate either a common evolutionary origin or the convergent evolution of features involved in RT function. Widely divergent telomerase RNAs, for example, share certain core secondary structures that are critical for telomerase activity but have no obvious counterparts in retroviral RNAs (5Lin J. Ly H. Hussain A. Abraham M. Pearl S. Tzfati Y. Parslow T.G. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14713-14718Crossref PubMed Scopus (89) Google Scholar, 6Chen J.L. Greider C.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14683-14684Crossref PubMed Scopus (75) Google Scholar). The single-stranded RNA genomes of retroviruses, on the other hand, share a tendency to form stable RNA homodimers that are found in all known retroviral particles and appear to be the natural substrates for viral RTs (reviewed in Ref. 7Paillart J.C. Shehu-Xhilaga M. Marquet R. Mak J. Nat Rev Microbiol. 2004; 2: 461-472Crossref PubMed Scopus (238) Google Scholar). The assembly of these viral genomic dimers is generally initiated at a specific palindrome (i.e. a self-complementary base sequence), unique to each virus, that dimerizes readily both in vitro and within cells, and mutations in these palindromes that disrupt genomic dimer formation can markedly impair viral replication and infectivity (7Paillart J.C. Shehu-Xhilaga M. Marquet R. Mak J. Nat Rev Microbiol. 2004; 2: 461-472Crossref PubMed Scopus (238) Google Scholar). RNAs derived from at least some cellular retroelements undergo similar dimerization (4Feng Y.X. Moore S.P. Garfinkel D.J. Rein A. J. Virol. 2000; 74: 10819-10821Crossref PubMed Scopus (39) Google Scholar). Unexpectedly, studies from our laboratory and others have recently shown that the human telomerase RNA also dimerizes readily and that this capacity to dimerize correlates with telomerase catalytic activity in cells or cell-free extracts (8Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar, 9Ly H. Xu L. Rivera M.A. Parslow T.G. Blackburn E.H. Genes Dev. 2003; 17: 1078-1083Crossref PubMed Scopus (46) Google Scholar). This raises the possibility that RNA dimerization plays a fundamental role in telomerase biology.In this study, we show that the telomerase RNA of Saccharomyces cerevisiae also forms dimers in vitro, although it otherwise bears little resemblance to its human counterpart. Yeast cells harboring a dimerization-defective allele of TLC1 exhibit a progressive telomere shortening effect over successive generations. Collectively, these results provide evidence that RNA template dimerization is a widely conserved property of reverse transcription in both cellular and retroviral contexts.EXPERIMENTAL PROCEDURESYeast Strain—Yeast strain yEHB1002 (MATα ade2::hisG his3Δ200 leu2Δ0 lys2D0 met15Δ0 trp1Δ63 ura3Δ0 tlc1:: TRP1pRS316TLC1) was constructed by disruption of the TLC1 gene with TRP1 (10Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 528-540Crossref PubMed Scopus (136) Google Scholar) (a generous gift of Dr. Elizabeth Blackburn at University of California, San Francisco). The strain carries a CEN/ARS, URA3 plasmid containing the wild-type TLC1 with its endogenous promoter and terminator sequence.In Vitro TLC1 RNA Dimerization Assay—RNAs were transcribed directly from PCR products containing either the full-length wild-type TLC1 sequence or various mutated or truncated versions of this gene using the MEGAscript kit as suggested by the manufacturer (Ambion, Austin, TX). TLC1 RNA was first denatured in a total of 8 μl of diethylpyrocarbonate water at 95 °C for 3 min and then immediately snap-cooled on ice for an additional 3 min. Two μl of 5× dimerization buffer (250 mm NaCl, 123 mm Tris (pH 7.0), 50 mm MgCl2) was added to each sample. The samples were either incubated on ice (0 °C) or at 37 °C for 120 min and then separated by nondenaturing gel electrophoresis in 90 mm Tris borate, 0.1 mm MgCl2. The percentage of RNA dimerization (% dimers) was calculated by dividing the intensity of the dimeric RNA band, as judged by NIH Image software, to the sum of both monomeric and dimeric RNA species in each lane.Southern Blot Analysis of Telomeres—Various TLC1 mutants created on the pRS313TLC1 plasmid were transformed into yeast strain yEHB1002 using the method described previously (5Lin J. Ly H. Hussain A. Abraham M. Pearl S. Tzfati Y. Parslow T.G. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14713-14718Crossref PubMed Scopus (89) Google Scholar). In some instances, two separate pRS313TLC1 plasmids carrying either the 42C or 42G mutant were co-transformed into yEHB1002 cells. Transformants were selected on agar plates that contained –Ura–His medium to retain both wild-type and mutant plasmids. Individual colonies were then streaked on plates that contain 5-flouroorotic acid-His to select against the wild-type copy of TLC1. Colonies were subsequently streaked on –His plate. Genomic DNA was isolated from cells at each streak, restriction digested with XhoI, separated on 0.8% agarose/Tris borate-EDTA, and transferred onto Nytran membrane (Schleicher & Schuell BioScience). The membrane was hybridized with a γ-32P-end-labeled probe, (TGTGGG)4, and visualized using a phosphorimaging technique (GE Healthcare).Northern Blot Analysis of TLC1—Total cellular RNAs (15 μg) isolated from yeast cells (yeast RNA purification system, Puregene, Minneapolis, MN) that carried either the wild-type copy or the different mutant versions of TLC1 and 1 ng of the in vitro transcribed 1,300-nt TLC1 RNA were treated with glyoxal prior to loading into a 1% agarose gel. After separation, the RNAs were transferred onto the Hybond membrane (Schleicher & Schuell Bioscience), UV-cross-linked, and hybridized to a TLC1 probe that was labeled by random priming (Amersham Biosciences) with [α-32P]dCTP. RNA markers (0.16–1.77 and 0.24–9.5 kb) purchased from Invitrogen were also similarly treated but were visualized by a methylene blue staining method (11Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual.3rd Ed. Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 7.36-7.39Google Scholar). The radiolabeled TLC1 RNAs on the membrane were exposed onto a phosphorimaging screen and analyzed. The membrane was stripped and rehybridized to a randomly primed probe corresponding to the yeast Tubulin-1 gene (a kind gift from Dr. Anita Corbett, Emory University) and visualized via a phosphorimaging technique (GE Healthcare).TLC1 Mutagenesis and Sequencing—Palindromic sequences located within the 3′ region of TLC1 were mutated by PCR mutagenesis (QuikChange mutagenesis kit, Strategene, La Jolla, CA) using pRS313 as the DNA template and primer pairs that contained the desired nucleotide substitutions or deletions. Similar strategy was used to replace the palindromic sequence of the S. cerevisiae with those of the other yeast strains shown in Fig. 4 (boxed sequences). Potential mutants, selected and sequenced in both directions to verify the desired changes, were used as templates for RNA transcription and for transformation into yeast cells. Plasmids containing various TLC1 sequences were isolated from yeast cells using the plasmid DNA purification system (Puregene, Minneapolis, MN) and sequenced. In the instance in which two plasmids expressing the dimerization complementation mutations were extracted from a single yeast cell, the mixture of the plasmids was extracted and transformed (by electroporation) into Escherichia coli. Plasmid DNAs were prepared using the Qiagen Miniprep kit (Qiagen) and submitted for sequencing to verify the identity of each of the dimerization mutant and that no inadvertent recombination had occurred between the two constructs.RESULTSNumerous reports demonstrate that synthetic transcripts representing specific portions of human telomerase RNA or of retroviral genomes dimerize spontaneously in vitro under near physiologic conditions (8Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar, 9Ly H. Xu L. Rivera M.A. Parslow T.G. Blackburn E.H. Genes Dev. 2003; 17: 1078-1083Crossref PubMed Scopus (46) Google Scholar, 12Moriarty T.J. Marie-Egyptienne D.T. Autexier C. Mol. Cell. Biol. 2004; 24: 3720-3733Crossref PubMed Scopus (96) Google Scholar, 13Clever J.L. Wong M.L. Parslow T.G. J. Virol. 1996; 70: 5902-5908Crossref PubMed Google Scholar, 14Laughrea M. Jette L. Biochemistry. 1994; 33: 13464-13474Crossref PubMed Scopus (222) Google Scholar, 15Marquet R. Paillart J.C. Skripkin E. Ehresmann C. Ehresmann B. Nucleic Acids Res. 1994; 22: 145-151Crossref PubMed Scopus (105) Google Scholar, 16Paillart J.C. Marquet R. Skripkin E. Ehresmann C. Ehresmann B. Biochimie (Paris). 1996; 78: 639-653Crossref PubMed Scopus (117) Google Scholar). In this study, we evaluated a synthetic 1300-nt S. cerevisiae TLC1 RNA under these standard conditions using an electrophoretic mobility shift assay (EMSA) and found that it readily formed dimer-sized complexes when warmed to 37 °C (Fig. 1B, lanes 2 and 3). Systematic truncation further revealed that a 416-nt TLC1 fragment comprising nts 885–1301 alone formed dimers at even higher efficiency than the full-length molecule (20–60% versus 15–20%), whereas isolated sequences from farther upstream did not form dimers efficiently (compare Fig. 1B, lanes 7 and 8 with lanes 5, 6, 9, and 10). When this 416-nt 3′ fragment and full-length TLC1 were combined in equal amounts (Fig. 1C, lanes 1–6, 18, and 19), we observed an RNA species migrating at the position predicted for a heterodimer of these two RNAs (double asterisks). Indeed, such heterodimeric RNA species appeared reproducibly to form more readily than would be expected for homodimers of the individual RNA species (Fig. 1C, single asterisks), perhaps because of reduced conformational variability or reduced steric effects on the truncated RNA. These findings confirm that the slow migrating complexes observed on EMSA are indeed dimers, and they suggest that specific sequences within the 3′ region of TLC1 are necessary and sufficient for dimer formation in vitro.FIGURE 1Sequences located within the 3′ region of TLC1 are necessary and sufficient to mediate RNA dimerize in vitro. A, schematic representation of the full-length TLC1 RNA (FL; 1300 nts), 5′ RNA fragment (450 nts), essential domain (ED) fragment (430 nts), and 3′ RNA fragment (416 nts). B, in vitro dimerizations of the full-length and truncated TLC1 RNAs. The percentage of RNA dimerization (% dimers) is a ratio of the amount of dimeric RNA to the sum of both monomeric and dimeric RNA species in each lane. Lanes 1 and 4, 0.24–9.5-kb RNA size ladder (Invitrogen) (D, dimers; M, monomers). C, EMSA showing efficient formations of the homodimers (single asterisks) and heterodimers (double asterisks) between RNA fragments of the wild-type (WT) sequence (lanes 1–6 and 18–19) or between RNA fragments containing either the 42C or the 42G mutation (lanes 7–16 and 20 and 21). Although only a small fraction of the heterodimers can be observed between the 42C- and 42G-containing RNA sequences at 2 h of incubation (lane 15), heterodimers form more readily after 4 h (lane 16) (double asterisk). The percentage of RNA dimerization is a ratio of the amount of dimeric RNA (either homodimer or heterodimer) to the sum of both monomeric and dimeric RNA species in each lane. L1, 0.16–1.77-kb RNA ladder (Invitrogen); L2, 0.24–9.5-kb RNA ladder (Invitrogen).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The signals that mediate dimerization of retroviral genomic RNAs typically include a sequence palindrome, 6 or more bases in length, that is critical for the initial interaction between RNA strands. We identified five such palindromes in the 3′ TLC1 region and created a series of mutants of this truncated RNA each of which introduced two or three nucleotide substitutions into a given palindrome (Fig. 2A). As shown in Fig. 2B, most of these mutations had no discernible effect on in vitro dimer formation as compared with the wild-type RNA species, with the exception of mutants 42C and 42G, which replaced the native palindrome CGCGCG at positions 1204–1209 with the alternative, non-palindromic sequences CCCCCG and CGGGGG, respectively (Fig. 1C, base substitutions are underlined). Each of the two latter mutations reduced dimerization to nearly undetectable levels (Figs. 1C, lanes 7–13, and 2B, lanes 5 and 6). Further evidence of a unique role for this palindrome was obtained by progressively truncating the 3′ end of the RNA (Fig. 2C). As shown in Fig. 2D, we found that RNAs that lacked up to 88 nt but retained this palindrome dimerized relatively efficiently (lanes 5–10), but that further deletions or base substitutions that eliminated the palindrome abolished dimer formation (lanes 1–4, 11, and 12).FIGURE 2CG-rich palindromic sequence located at positions 1204–1209 in the 3′ RNA fragment is required for TLC1 dimerization. A, schematic representation of the locations of targeted mutagenesis of the 6-base palindromic sequences located within the 3′ RNA fragment. The altered nucleotides are underlined. B, EMSA shows defective dimerization of the mutant 42G (lanes 5 and 6) as compared with either the wild type (lanes 1 and 2) or other mutated sequences (lanes 3 and 4 and 7–12). 0.16–1.77-kb RNA ladder. C, schematic representation of various RNA species generated from DNA templates that terminate at the indicated enzymatic restriction sites and positions. D, EMSA showing that whereas all RNA fragments that contain the wild-type CG-rich palindromic sequence(WT) at position 1204–1209 (blackboxes) can dimerize efficiently, those that carry either the mutated palindrome or lack this sequence completely (lanes 1–4) fail to form dimers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The significance of palindromes in dimer initiation reflects their ability to mediate homologous base-pairing interactions between RNA strands. The 42C and 42G mutations replace the native palindrome at positions 1204–1209 with sequences that are nonpalindromic but are complementary to each other and so might be predicted to support heterodimer formation. We tested this possibility by combining a full-length TCL1 that contained the 42G mutation with an equimolar amount of a 416-nt 3′ fragment containing 42C. As shown in Fig. 1C, neither of these mutant RNAs formed appreciable homodimers when incubated alone for either 2 h (lanes 7, 8, 10–13, 20, and 21) or up to 4 h (data not shown), but small amounts of an apparent heterodimer reproducibly formed when the two RNA species were mixed (lanes 15 and 16). Formation of this mutant heterodimer, however, was slow and inefficient in comparison with homodimerization of wild-type TLC1 (Fig. 1C, compare lane 6 with lanes 15 and 16); this is consistent with evidence from retroviral systems that only a small subset of complementary sequences can support dimerization in vitro or in vivo (13Clever J.L. Wong M.L. Parslow T.G. J. Virol. 1996; 70: 5902-5908Crossref PubMed Google Scholar, 17Laughrea M. Jette L. Mak J. Kleiman L. Liang C. Wainberg M.A. J. Virol. 1997; 71: 3397-3406Crossref PubMed Google Scholar, 18Laughrea M. Jette L. Biochemistry. 1997; 36: 9501-9508Crossref PubMed Scopus (53) Google Scholar). Taken together, these results indicate that the palindrome at positions 1204–1209 is required for efficient TLC1 dimer formation in vitro by virtue of its ability to form base pairs between strands.We next asked whether TLC1 dimerization plays a role in the life cycle of yeast cells. To do this, we created artificial expression cassettes encoding wild-type or mutant forms of the full-length TLC1 sequence and introduced these in place of the endogenous TLC1 gene in S. cerevisiae (as outlined under “Experimental Procedures”). Individual transformed yeast clones with the desired genotypes, verified by DNA sequencing, were selected and characterized further by continuous cultivation (i.e. by streaking the same population of cells on agar plates for five successive passages). As shown in Fig. 3A, yeast strains that carried only the wild-type TLC1 allele or the dimerization-competent control mutant 43 grew at roughly comparable rates. In contrast, cells harboring either of the dimerization-defective mutants (42C or 42G) alone grew somewhat more slowly and were less efficiently propagated. It is important to note, however, that neither mutant entered replicative senescence, even by the fifth passage, highlighting the plasticity of yeast cells in cultured conditions. Compound heterozygous cells that contained both the 42C and 42G alleles appeared to grow better than those with either mutant alone (Fig. 3A). Similar results were obtained when similar strains of yeast cells were grown in liquid cultures prior to being serially diluted and spotted onto plates for growth (Fig. 3A, 1–4). These results suggest that the 42C and 42G mutants can at least partially complement each other functionally in trans, presumably because of their ability to form RNA heterodimers.FIGURE 3Yeast cells expressing different versions of the TLC1 dimerization-defective RNA grow more slowly and possess shorter telomeres than those that express the dimerization competent TLC1 molecules. Yeast cells harboring either the wild-type TLC1 RNA, dimerization-defective mutants 42G and 42C (either alone or together) or a dimerization-competent palindromic mutant (Mut 43) were passaged serially by continuous streaking onto fresh agar plates. A, by streak 3, compared with cells that carry either the wild-type TLC1 or Mut 43, those that carry either of the dimerization-defective mutants (42G or 42G) seem to exhibit a slower growth rate, a phenotype that seems to be rescued in cells that carry both versions of the dimerization-defective mutants. At streak 5 (∼100 generations), representative yeast liquid cultures containing the 42C+42G compound heterozygous mutants (1Blackburn E.H. Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1750) Google Scholar), 42G alone (2Lingner J. Hughes T.R. Shevchenko A. Mann M. Lundblad V. Cech T.R. Science. 1997; 276: 561-567Crossref PubMed Scopus (1032) Google Scholar), 42C alone (3Peng Y. Mian I.S. Lue N.F. Mol. Cell. 2001; 7: 1201-1211Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), or the wild-type TLC1 (4Feng Y.X. Moore S.P. Garfinkel D.J. Rein A. J. Virol. 2000; 74: 10819-10821Crossref PubMed Scopus (39) Google Scholar) were grown to A600 = 1.0, serially diluted by 1:3 (five serial dilutions), spotted onto a plate, and grown at 30 °C for 3 days. B, Southern blotting analysis of the telomeric DNAs isolated from yeast cells expressing either the wild-type (WT) copy (lanes 1, 2, 9, and 10) or the mutant 43 copy of the TLC1 (lanes 6–8) shows constant telomere length maintenance over successive passaging (streaks 1–5) but progressive telomere shortening effects in cells that express either of the dimerization-defective mutants (lanes 3–5 and 14–18). In contrast, cells that express both copies of the dimerization-defective mutants 42C and 42G (lanes 12 and 13) maintain telomere lengths at levels similar to those that express either the wild-type or mutant 43 copy, suggesting that both mutants 42C and 42G function in trans to restore normal telomerase activity. Lane 11 is empty. C, Northern blotting analysis shows that the dimerization-defective mutants 42G and 42C possess levels of RNA stability similar to that of the wild-type fragment (lanes 1–3 and 7–9). The membrane was first probed for the 1300-nt TLC1 RNA (lanes 1–3) and then reprobed for the 1600-nt cellular α-tubulin-1 RNA (lanes 1′–3′). The 0.24–9.5-kb RNA ladder (Invitrogen) was separated on the same gel and similarly transferred onto the membrane before being stained with methylene blue dye. In vitro transcribed TLC1 RNAs representing the mature 1300-nt form (at 1 ng and 10 ng in lanes 4 and 5, respectively) were separated on the same gel as those RNAs extracted directly from yeast cells (lanes 7–9). Lane 6 is empty.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Further evidence of a possible functional role for TLC1 dimerization was obtained by examining telomere lengths of these same yeast strains by Southern blot analysis (Fig. 3B). Over successive passages, telomere length decreased progressively in yeast that carried the dimerization-defective mutant 42C or 42G alone, whereas it remained roughly constant in yeast carrying wild-type TLC1 or the control dimerization-efficient mutant 43. Despite their abnormal lengths, telomeres in the 42C and 42G single-mutant strains remained stable through five passages, consistent with the nonsenescent phenotype we observed. Moreover, heterozygous yeast expressing the 42C and 42G alleles together maintained roughly normal telomere lengths, again suggesting phenotypic trans-complementation by these two mutant alleles. Collectively, these data are compatible with the view that interstrand base-pairing interactions involving the palindromic residues 1204–1209 of TLC1 may be required for maintenance of optimal telomere length in yeast cells.Efforts to map the 3′ end of yeast TLC1 RNA in vivo have produced conflicting results (19Chapon C. Cech T.R. Zaug A.J. RNA. 1997; 3: 1337-1351PubMed Google Scholar, 20Chappell A.S. Lundblad V. Mol. Cell. Biol. 2004; 24: 7720-7736Crossref PubMed Scopus (43) Google Scholar, 21Seto A.G. Zaug A.J. Sobel S.G. Wolin S.L. Cech T.R. Nature. 1999; 401: 177-180Crossref PubMed Scopus (230) Google Scholar, 22Singer M.S. Gottschling D.E. Science. 1994; 266: 404-409Crossref PubMed Scopus (649) Google Scholar, 23Dandjinou A.T. Levesque N. Larose S. Lucier J.F. Abou Elela S. Wellinger R.J. Curr. Biol. 2004; 14: 1148-1158Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 24Bosoy D. Peng Y. Mian I.S. Lue N.F. J. Biol. Chem. 2003; 278: 3882-3890Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Zappulla D.C. Goodrich K. Cech T.R. Nat. Struct. Mol. Biol. 2005; 12: 1072-1077Crossref PubMed Scopus (67) Google Scholar). Although some investigators report evidence to suggest that the terminal nucleotide of TLC1 maps at or near position 1250, which would include the dimer-initiating palindromic sequence identified in our study (19Chapon C. Cech T.R. Zaug A.J. RNA. 1997; 3: 1337-1351PubMed Google Scholar, 20Chappell A.S. Lundblad V. Mol. Cell. Biol. 2004; 24: 7720-7736Crossref PubMed Scopus (43) Google Scholar, 21Seto A.G. Zaug A.J. Sobel S.G. Wolin S.L. Cech T.R. Nature. 1999; 401: 177-180Crossref PubMed Scopus (230) Google Scholar, 22Singer M.S. Gottschling D.E. Science. 1994; 266: 404-409Crossref PubMed Scopus (649) Google Scholar), other studies indicate that TLC1 RNA may end at or near position 1167 (Fig. 2C) (23Dandjinou A.T. Levesque N. Larose S. Lucier J.F. Abou Elela S. Wellinger R.J. Curr. Biol. 2004; 14: 1148-1158Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 24Bosoy D. Peng Y. Mian I.S. Lue N.F. J. Biol. Chem. 2003; 278: 3882-3890Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Zappulla D.C. Goodrich K. Cech T.R. Nat. Struct. Mol. Biol. 2005; 12: 1072-1077Crossref PubMed Scopus (67) Google Scholar). A recent study performed by Dandjinou et al. (23Dandjinou A.T. Levesque N. Larose S. Lucier J.F. Abou Elela S. Wellinger R.J. Curr. Biol. 2004; 14: 1148-1158Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), however, has confirmed the presence of both RNA species in yeast cells and has suggested that the region downstream of position 1167 might contribute to either RNA stability or other aspects of telomerase RNP function such as dimerization or multimerization. In the current study, we confirmed by Northern blot (Fig. 3C) that the wild type and each of the dimerization-defective mutants, 42C and 42G, generated stable RNA products that had lengths comparable with the in vitro transcribed 1300-nt TLC1 RNA (lanes 4 and 5).Finally, we searched for functional counterparts of the dimerization-directed palindrome in TLC1 sequences from Saccharomyces cariocanus and Saccharomyces kudriavzevii, two other yeast species that belong to the senso stricto group and in which TLC1 RNAs exhibit 77 and 65% sequence identity overall, respectively, to that of S. cerevisiae (23Dandjinou A.T. Levesque N. Larose S. Lucier J.F. Abou Elela S. Wellinger R.J. Curr. Biol. 2004; 14: 1148-1158Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). When these sequences are optimally aligned, the regions in question are markedly divergent among the three species but are either palindromic (in the case of S. kudriavzevii) or imperfectly palindromic (in the case of the S. cariocanus) to a degree that would still allow base pairing to occur (Fig. 4A, boxed). As full-length clones of the S. cariocanus and S. kudriavzevii TLC1 genes were not available to us, we were unable to evaluate the dimerization properties of the native RNAs; instead, we asked whether the palindromic sequences from those genes could functionally replace residues 1204–1209 in the context of S. cerevisiae TLC1. Fig. 4B demonstrates that dimerization of the S. cerevisiae RNA was completely abolished when the native palindrome was deleted but could be restored to essentially full efficiency by inserting either the S. cariocanus or the S. kudriavzevii palindrome in its place. Thus, despite wide sequence divergence over the evolutionary history of these species, local palindromic character has been conserved that" @default.
• W2009099360 created "2016-06-24" @default.
• W2009099360 creator A5014083162 @default.
• W2009099360 creator A5014611789 @default.
• W2009099360 creator A5030145353 @default.
• W2009099360 creator A5035768241 @default.
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• W2009099360 date "2007-06-01" @default.
• W2009099360 modified "2023-10-18" @default.
• W2009099360 title "Functional Characterization of Yeast Telomerase RNA Dimerization" @default.
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