Assessing Telomeric Phenotypes

LeBel, Catherine; Larrivée, Michel; Bah, Amadou; Laterreur, Nancy; Lvesque, Nancy; Wellinger, Raymund J.

doi:10.1385/1-59259-958-3:265

Cited by 7 publications

(11 citation statements)

References 51 publications

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“…DNA was extracted from relevant strains and digested with the restriction enzyme XhoI. Due to a conserved XhoI site in the telomere-proximal YЈ-elements, many terminal restriction fragments appear as somewhat smeary bands of about 1.2 kb size on such blots (44). In the absence of Rnt1p, the size of the terminal restriction fragments increased as compared with that of wild type cells, indicating that the deletion of RNT1 increases telomere length ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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RNase III-dependent Regulation of Yeast Telomerase

Larose

Laterreur

Ghazal

et al. 2007

Journal of Biological Chemistry

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In bakers' yeast, in vivo telomerase activity requires a ribonucleoprotein (RNP) complex with at least four associated proteins (Est2p, Est1p, Est3p, and Cdc13p) and one RNA species (Tlc1). The function of telomerase in maintaining chromosome ends, called telomeres, is tightly regulated and linked to the cell cycle. However, the mechanisms that regulate the expression of individual components of telomerase are poorly understood. Here we report that yeast RNase III (Rnt1p), a double-stranded RNA-specific endoribonuclease, regulates the expression of telomerase subunits and is required for maintaining normal telomere length. Deletion or inactivation of RNT1 induced the expression of Est1, Est2, Est3, and Tlc1 RNAs and increased telomerase activity, leading to elongation of telomeric repeat tracts. In silico analysis of the different RNAs coding for the telomerase subunits revealed a canonical Rnt1p cleavage site near the 3 end of Est1 mRNA. This predicted structure was cleaved by Rnt1p and its disruption abolished cleavage in vitro. Mutation of the Rnt1p cleavage signal in vivo impaired the cell cycle-dependent degradation of Est1 mRNA without affecting its steady-state level. These results reveal a new mechanism that influences telomeres length by controlling the expression of the telomerase subunits.The ends of eukaryotic chromosomes are capped with special structures made of tandem DNA repeats and associated proteins, called telomeres. These structures protect chromosomes from end-to-end fusion, recombination, and nucleolytic degradation (1, 2). However, because the conventional DNA replication machinery cannot fully duplicate the ends of linear chromosomes, telomeric DNA will shorten with each round of replication, leading to a replicative senescence (3). To solve this end replication problem, most eukaryotes use the activity of an enzyme called telomerase to ensure the maintenance of telomeric DNA (4). Telomerase is a ribonucleoprotein reverse transcriptase that can extend the 3Ј end of chromosomes using its RNA subunit as a template for the addition of telomeric repeats.In vitro, telomerase activity requires a reverse transcriptase (Tert, Est2p in yeast) and an RNA template (Terc, Tlc1 in yeast)

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Section: Resultsmentioning

confidence: 99%

“…In this procedure, telomerase was partially purified using IgG-Sepharose beads and used for a direct extension of a telomeric primer (44). As shown in Fig.…”

Section: Rnt1 Rnt1⌬mentioning

confidence: 99%

RNase III-dependent Regulation of Yeast Telomerase

Larose

Laterreur

Ghazal

et al. 2007

Journal of Biological Chemistry

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“…32 P-radiolabeled probes were used to detect single-stranded and double-stranded telomeric DNA containing either vertebrate-or yeast-specific repeats in XhoI-digested genomic DNAs resolved on agarose gels, essentially as described previously (23,31).…”

Section: Methodsmentioning

confidence: 99%

Telomerase Is Required to Protect Chromosomes with Vertebrate-type T2AG3 3′ Ends in Saccharomyces cerevisiae

Bah

Gilson

Wellinger

2011

Journal of Biological Chemistry

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Telomeres containing vertebrate-type DNA repeats can be stably maintained in Saccharomyces cerevisiae cells. We show here that telomerase is required for growth of yeast cells containing these vertebrate-type telomeres. When present at the chromosome termini, these heterologous repeats elicit a DNA damage response and a certain deprotection of telomeres. The data also show that these phenotypes are due only to the terminal localization of the vertebrate repeats because if they are sandwiched between native yeast repeats, no phenotype is observed. Indeed and quite surprisingly, in this latter situation, telomeres are of virtually normal lengths, despite the presence of up to 50% of heterologous repeats. Furthermore, the presence of the distal vertebrate-type repeats can cause increased problems of the replication fork. These results show that in budding yeast the integrity of the 3 overhang is required for proper termination of telomere replication as well as protection.Specialized ribonucleoprotein complexes known as telomeres protect the natural ends of eukaryotic chromosomes. In the budding yeast Saccharomyces cerevisiae, telomeres are composed of an ϳ300-bp double-stranded (ds) 4 tract of irregular repeats, abbreviated TG 1-3 /C 1-3 A, and terminate with a 12-to 14-nt single-stranded (ss) 3Ј overhang made of the TG 1-3 motifs (1-3). In contrast, vertebrate telomeres contain a ds tract containing thousands of T 2 AG 3 /C 3 TA 2 repeats and terminate with a ss 3Ј overhang made of T 2 AG 3 motifs (2, 4, 5).The complete replication of linear eukaryotic chromosomes requires the de novo addition of telomeric repeats to chromosome ends by telomerase, a specialized reverse transcriptase (for reviews, see Refs. 6 -9). The core telomerase components are a reverse-transcriptase catalytic subunit and a RNA moiety. In yeast, these components are known as Est2p and TLC1, respectively (8). The telomerase RNA always contains a short region that is complementary to the G-rich strand of telomeric repeats, and that region is used as a template for repeat addition (7, 9).Telomerase invalidation leads to gradual telomere shortening and ultimately causes cells to stop dividing (7, 9, 10). Nevertheless, cellular backup mechanisms can maintain telomeres in the absence of telomerase and ultimately preserve genome integrity. In this mode of telomere maintenance, which is known as alternative lengthening of telomeres in human cells, and survivor mode in Saccharomyces cerevisiae, telomeric DNA is maintained via homologous recombination-based mechanisms (11,12).By differentiating chromosomal ends from internal ds breaks, telomeres also prevent chromosomal ends from eliciting DNA damage checkpoint activation and protect telomeres from inappropriate DNA repair activity. Binding of the multiprotein complex shelterin is essential for both protecting and maintaining the length of mammalian telomeres (13,14). Rap1p, one of the several proteins associated with similar functions at budding yeast telomeres, binds directly to ds telomeric repeats ...

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“…Senescence assays on plates were performed according to Maringele and Lydall (2004) and Lebel et al (2006). Briefly, cells were isolated from freshly dissected tetrads (passage 1) and propagated on YPD plates.…”

Section: Methodsmentioning

confidence: 99%

“…The membrane was hybridized to a radiolabeled yeast telomeric oligo (59-CACACCCACACCCACACC-39) to detect terminal restriction fragments (TRFs) (Lebel et al 2006). As a loading control, a 1.76-kbp fragment of the CDC15 locus was amplified as described in Foster et al (2006) and Zubko and Lydall (2006), labeled, and used to probe genomic DNA digested with XhoI to reveal a single hybridization fragment at $3 kbp.…”

Section: Methodsmentioning

confidence: 99%

Telomere Maintenance and Survival in Saccharomyces cerevisiae in the Absence of Telomerase and RAD52

et al. 2009

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Telomeres are essential features of linear genomes that are crucial for chromosome stability. Telomeric DNA is usually replenished by telomerase. Deletion of genes encoding telomerase components leads to telomere attrition with each cycle of DNA replication, eventually causing cell senescence or death. In the Saccharomyces cerevisiae strain W303, telomerase-null populations bypass senescence and, unless EXO1 is also deleted, this survival is RAD52 dependent. Unexpectedly, we found that the S. cerevisiae strain S288C could survive the removal of RAD52 and telomerase at a low frequency without additional gene deletions. These RAD52-independent survivors were propagated stably and exhibited a telomere organization typical of recombination between telomeric DNA tracts, and in diploids behaved as a multigenic trait. The polymerase-d subunit Pol32 was dispensable for the maintenance of RAD52-independent survivors. The incidence of this rare escape was not affected by deletion of other genes necessary for RAD52-dependent survival, but correlated with initial telomere length. If W303 strains lacking telomerase and RAD52 first underwent telomere elongation, rare colonies could then bypass senescence. We suggest that longer telomeres provide a more proficient substrate for a novel telomere maintenance mechanism that does not rely on telomerase, RAD52, or POL32.

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Assessing Telomeric Phenotypes

Cited by 7 publications

References 51 publications

RNase III-dependent Regulation of Yeast Telomerase

RNase III-dependent Regulation of Yeast Telomerase

Telomerase Is Required to Protect Chromosomes with Vertebrate-type T2AG3 3′ Ends in Saccharomyces cerevisiae

Telomere Maintenance and Survival in Saccharomyces cerevisiae in the Absence of Telomerase and RAD52

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