Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment

Replicative Senescence ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Telomeric Dna ◽

In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished—for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 by increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab360 ◽

2021 ◽

Author(s):

Enikő Fekete-Szücs ◽

Fernando R Rosas Bringas ◽

Sonia Stinus ◽

Michael Chang

Keyword(s):

Replicative Senescence ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Telomeric Dna ◽

Abstract In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished—for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 by increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915312116 ◽

2019 ◽

Vol 116 (49) ◽

pp. 24542-24550 ◽

Cited By ~ 8

Author(s):

Jiarui Song ◽

Dhenugen Logeswaran ◽

Claudia Castillo-González ◽

Yang Li ◽

Sreyashree Bose ◽

...

Keyword(s):

Telomerase Activity ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Telomeric Dna ◽

Structural Element ◽

Stem Loop ◽

Evolutionary Pathway ◽

Pol Iii

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

Essential Regions of Saccharomyces cerevisiae Telomerase RNA: Separate Elements for Est1p and Est2p Interaction

Molecular and Cellular Biology ◽

10.1128/mcb.22.7.2366-2374.2002 ◽

2002 ◽

Vol 22 (7) ◽

pp. 2366-2374 ◽

Cited By ~ 70

Author(s):

April J. Livengood ◽

Arthur J. Zaug ◽

Thomas R. Cech

Keyword(s):

Nucleotide Sequence ◽

Dna Synthesis ◽

Active Site ◽

Telomerase Rna ◽

Telomeric Dna ◽

Protein Subunits ◽

Essential Region ◽

Selection For

ABSTRACT The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (∼0.5 kb of the ∼1.3-kb RNA) is required for telomerase function in vivo. The main essential region (430 nucleotides), which contains the template for telomeric DNA synthesis, was required for coimmunoprecipitation with Est1p and Est2p. Furthermore, the subregion required for interaction with Est1p, the telomerase recruitment subunit, differed from those required for interaction with Est2p, the reverse transcriptase subunit. Two regions of the RNA distant from the template in the nucleotide sequence were required for Est2p binding, but the template itself was not. Having the RNA secured to the protein away from the template is proposed to facilitate the translocation of the RNA template through the active site. More generally, our results support a role for the telomerase RNA serving as a scaffold for binding key protein subunits.

Repositioning the Sm-Binding Site in Saccharomyces cerevisiae Telomerase RNA Reveals RNP Organizational Flexibility and Sm-Directed 3′-End Formation

Non-Coding RNA ◽

10.3390/ncrna6010009 ◽

2020 ◽

Vol 6 (1) ◽

pp. 9 ◽

Cited By ~ 1

Author(s):

Evan P. Hass ◽

David C. Zappulla

Keyword(s):

Binding Site ◽

Telomerase Rna ◽

Telomeric Dna ◽

Protein Subunits ◽

Yeast Telomerase ◽

Rna Biogenesis ◽

Native Site ◽

Processing Mechanism

Telomerase RNA contains a template for synthesizing telomeric DNA and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase RNA, TLC1, is bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. However, it remains unclear (1) whether Sm7 retains this function when its binding site is repositioned within TLC1, as has been shown for other TLC1-binding telomerase subunits, and (2) how Sm7 stabilizes poly(A)– TLC1. Here, we first show that Sm7 can stabilize poly(A)– TLC1 even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we show that when an Sm site is inserted 5′ of its native position and the native site is mutated, Sm7 stabilizes shorter forms of poly(A)– TLC1 in a manner corresponding to how far upstream the new site was inserted, providing strong evidence that Sm7 binding to TLC1 controls where the mature poly(A)– 3′ is formed by directing a 3′-to-5′ processing mechanism. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

Ty1 Mobilizes Subtelomeric Y′ Elements in Telomerase-Negative Saccharomyces cerevisiae Survivors

Molecular and Cellular Biology ◽

10.1128/mcb.24.22.9887-9898.2004 ◽

2004 ◽

Vol 24 (22) ◽

pp. 9887-9898 ◽

Cited By ~ 30

Author(s):

Patrick H. Maxwell ◽

Candice Coombes ◽

Alison E. Kenny ◽

Joseph F. Lawler ◽

Jef D. Boeke ◽

...

Keyword(s):

Transcription Factor ◽

Cell Division ◽

Long Terminal Repeat ◽

Reverse Transcription ◽

Telomere Maintenance ◽

Nonpermissive Temperature ◽

Telomeric Dna ◽

Copy Numbers ◽

Ty1 Retrotransposition

ABSTRACT When telomerase is inactivated in Saccharomyces cerevisiae, telomeric DNA shortens with every cell division, and cells stop dividing after ∼100 generations. Survivors that form in these senescent populations and resume growing have variably amplified arrays of subtelomeric Y′ elements. We marked a chromosomal Y′ element with the his3AI retrotransposition indicator gene and found that Y′HIS3 cDNA was incorporated into the genome at ∼10- to 1,000-fold-higher frequencies in survivors compared to telomerase-positive strains. Y′HIS3 cDNA mobility was significantly reduced if assayed at 30°C, a nonpermissive temperature for Ty1 retrotransposition, or in the absence of Tec1p, a transcription factor for Ty1. Microarray analysis revealed that Y′ RNA is preferentially associated with Ty1 virus-like particles (VLPs). Genomic copies of Y′HIS3 cDNA typically have downstream oligo(A) tracts, followed by a complete Ty1 long terminal repeat and TYA1 or TYB1 sequences. These data are consistent with the use of Ty1 cDNA to prime reverse transcription of polyadenylated Y′ RNA within Ty1 VLPs. Unmarked Y′-oligo(A)-Ty1 cDNA was also detected in survivors, reaching copy numbers of ∼10−2 per genome. We propose that Y′-oligo(A)-Ty1 cDNA recombines with Y′ elements at eroding telomeres in survivors and may play a role in telomere maintenance in the absence of telomerase.

Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.4961 ◽

1998 ◽

Vol 18 (9) ◽

pp. 4961-4970 ◽

Cited By ~ 31

Author(s):

Tracy Boswell Fulton ◽

Elizabeth H. Blackburn

Keyword(s):

Kluyveromyces Lactis ◽

Telomerase Activity ◽

Reaction Products ◽

Telomeric Dna ◽

Whole Cell ◽

Cell Extracts ◽

Dna Template ◽

ABSTRACT Telomeres in the budding yeast Kluyveromyces lactisconsist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactistelomerase RNA, encoded by the TER1 gene, is ∼1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactiswhole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactistelomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactistelomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactistelomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.

Telomerase-Independent Proliferation Is Influenced by Cell Type in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/164.3.909 ◽

2003 ◽

Vol 164 (3) ◽

pp. 909-921

Author(s):

Joanna E Lowell ◽

Alexander I Roughton ◽

Victoria Lundblad ◽

Lorraine Pillus

Keyword(s):

Replicative Senescence ◽

Telomere Maintenance ◽

Cell Type ◽

Telomeric Dna ◽

Yeast Strains ◽

Cell Type Specific ◽

Specific Regulation ◽

Mat Locus ◽

Dependent Pathway

Abstract Yeast strains harboring mutations in genes required for telomerase function (TLC1 and the EST genes) exhibit progressive shortening of telomeric DNA and replicative senescence. A minority of cells withstands loss of telomerase through RAD52-dependent amplification of telomeric and subtelomeric sequences; such survivors are now capable of long-term propagation with telomeres maintained by recombination rather than by telomerase. Here we report that simultaneous expression in haploid cells of both MATa and MATα information suppresses the senescence of telomerase-deficient mutants, with suppression occurring via the RAD52-dependent survivor pathway(s). Such suppression can be mimicked by deletion of SIR1-SIR4, genes that function in transcriptional silencing of several loci including the silent mating-type loci. Furthermore, telomerase-defective diploid strains that express only MATa or MATα information senesce at a faster rate than telomerase-defective diploids that are heterozygous at the MAT locus. This suggests that the RAD52-dependent pathway(s) for telomere maintenance respond to changes in the levels of recombination, a process regulated in part by the hierarchy of gene control that includes MAT regulation. We propose that cell-type-specific regulation of recombination at human telomeres may similarly contribute to the tissue-specific patterns of disease found in telomerase-deficient tumors.

Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

10.1101/167361 ◽

2017 ◽

Cited By ~ 2

Author(s):

Evan P. Hass ◽

David C. Zappulla

Keyword(s):

Binding Site ◽

Strong Evidence ◽

Binding Sites ◽

Telomerase Rna ◽

Telomeric Dna ◽

Protein Subunits ◽

Yeast Telomerase ◽

Rna Biogenesis

ABSTRACTTelomerase RNA contains a template for synthesizing telomeric DNA by reverse transcription and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase subunits Est1 and Ku bind to the telomerase RNA, TLC1, and it has been shown that these proteins still function when their binding sites are repositioned within the RNA. TLC1 is also bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. Here, we first show that Sm7 can perform this function even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we tested the hypothesis that the location of the Sm7-binding site relative to the 3′ end is contrastingly important. When we moved the Sm site to locations 5′ of its native position, we observed that this stabilized shorter forms of poly(A)– TLC1 in a manner precisely corresponding to how far upstream the Sm site was moved. This provides strong evidence that the location of Sm7 binding to TLC1 controls where the mature poly(A)– 3′ end is formed. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

Either Rap1 or Cdc13 can protect telomeric single-stranded 3′ overhangs from degradation in vitro

Scientific Reports ◽

10.1038/s41598-019-55482-3 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Rikard Runnberg ◽

Saishyam Narayanan ◽

Humberto Itriago ◽

Marita Cohn

Keyword(s):

Genome Stability ◽

Replicative Senescence ◽

Repetitive Sequences ◽

Protein Structures ◽

Telomeric Dna ◽

Telomere Function ◽

Exonucleolytic Degradation ◽

Linear Chromosomes ◽

AbstractTelomeres, the DNA-protein structures capping the ends of linear chromosomes, are important for regulating replicative senescence and maintaining genome stability. Telomeres consist of G-rich repetitive sequences that end in a G-rich single-stranded (ss) 3′ overhang, which is vital for telomere function. It is largely unknown how the 3′ overhang is protected against exonucleases. In budding yeast, double-stranded (ds) telomeric DNA is bound by Rap1, while ssDNA is bound by Cdc13. Here, we developed an in vitro DNA 3′end protection assay to gain mechanistic insight into how Naumovozyma castellii Cdc13 and Rap1 may protect against 3′ exonucleolytic degradation by Exonuclease T. Our results show that Cdc13 protects the 3′ overhang at least 5 nucleotides (nt) beyond its binding site, when bound directly adjacent to the ds-ss junction. Rap1 protects 1–2 nt of the 3′ overhang when bound to dsDNA adjacent to the ds-ss junction. Remarkably, when Rap1 is bound across the ds-ss junction, the protection of the 3′ overhang is extended to 6 nt. This shows that binding by either Cdc13 or Rap1 can protect telomeric overhangs from 3′ exonucleolytic degradation, and suggests a new important role for Rap1 in protecting short overhangs under circumstances when Cdc13 cannot bind the telomere.

Expression of telomerase prevents ALT and maintains telomeric heterochromatin in juvenile brain tumors

10.1101/718551 ◽

2019 ◽

Cited By ~ 2

Author(s):

Aurora Irene Idilli ◽

Emilio Cusanelli ◽

Francesca Pagani ◽

Emanuela Kerschbamer ◽

Francesco Berardinelli ◽

...

Keyword(s):

Brain Tumor ◽

Brain Tumors ◽

Cancer Progression ◽

Replicative Senescence ◽

Telomere Maintenance ◽

Telomeric Dna ◽

Expression Of Genes ◽

Alternative Lengthening ◽

Brain Tumor Cells ◽

Telomeric Heterochromatin

ABSTRACTThe activation of a telomere maintenance mechanism (TMM) is an essential step in cancer progression to escape replicative senescence and apoptosis. Paediatric brain tumors frequently exhibit Alternative Lengthening of Telomere (ALT) as active TMM, but the mechanisms involved in the induction of ALT in brain tumor cells are not clear.Here, we report a model of juvenile zebrafish brain tumor that progressively develops ALT. We discovered that reduced expression of tert and increase in Terra expression precedes ALT development. Additionally, tumors show persistent telomeric DNA damage and loss of heterochromatin marks at chromosome ends. Surprisingly, expression of telomerase reverts ALT features. Comparative analysis of gene expression after the rescue of ALT with telomerase and analysis of telomerase positive paediatric brain cancers showed increase of telomeric heterochromatin and maintenance of telomere length compared to ALT tumors, with reduced expression of genes of the pre-replicative complex as hallmark. Thus our study identifies telomere maintenance mechanisms as major drivers of telomeric DNA replication and chromatin status in brain cancers.