The Est1 Subunit of Yeast Telomerase Binds the Tlc1 Telomerase RNA

ABSTRACT Est1 is a component of yeast telomerase, and est1mutants have senescence and telomere loss phenotypes. The exact function of Est1 is not known, and it is not homologous to components of other telomerases. We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity. Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1. Deletion of EBS1 results in short telomeres. We created point mutations in a putative RRM of Est1. One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants. Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA. Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA. Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM. Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.

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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):

Saccharomyces Cerevisiae ◽

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.

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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):

Saccharomyces Cerevisiae ◽

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.

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Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5658-5664 ◽

Cited By ~ 48

Author(s):

Soo-Jin Yang ◽

Nagendra N. Mishra ◽

Aileen Rubio ◽

Arnold S. Bayer

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Content Type ◽

Reading Frame ◽

A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

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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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3329 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3329-3340 ◽

Cited By ~ 59

Author(s):

Tara L. Beattie ◽

Wen Zhou ◽

Murray O. Robinson ◽

Lea Harrington

Keyword(s):

Telomerase Activity ◽

Amino Terminus ◽

Telomerase Rna ◽

Carboxy Terminus ◽

Active Core ◽

Human Telomerase ◽

Precise Role

The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.

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Comprehensive Structure-Function Analysis of the Core Domain of Human Telomerase RNA

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.6849-6856.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 6849-6856 ◽

Cited By ~ 62

Author(s):

Hinh Ly ◽

Elizabeth H. Blackburn ◽

Tristram G. Parslow

Keyword(s):

Mutational Analysis ◽

Function Analysis ◽

Telomerase Activity ◽

Telomerase Rna ◽

Dna Repeats ◽

Telomeric Dna ◽

Core Domain ◽

The Core ◽

Optimal Function ◽

Human Telomerase

ABSTRACT Telomerase is a cellular reverse transcriptase that uses part of its integral RNA (called TER) as the template to synthesize telomeric DNA repeats. Vertebrate TERs are thought to share a conserved, highly structured core domain that includes the templating sequence and a pseudoknot, but not all features of the predicted core structure have been verified directly or shown to affect telomerase enzymatic activity. Here, we report a systematic mutational analysis of the core domain (residues 1 to 210) of human telomerase RNA (hTER). Our data confirm that optimal hTER activity requires the integrity of four short helices (P2a.1, P2a, P2b, and P3) which create the proposed pseudoknot and that features of both the primary sequence and secondary structure in P2b and P3 contribute to optimal function. At least part of the long-range P1 pairing is also required, despite the lack of a known P1 counterpart in rodent TERs. Among the predicted single-stranded regions, we found that J2b/3, portions of J2a/3, and residues in and around the template make sequence-specific contributions to telomerase function. Additionally, we provide evidence that naturally occurring hTER sequence polymorphisms found in some patients with aplastic anemia can inhibit telomerase activity by disrupting critical structures within the hTER core domain.

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A 4-base pair core-enclosing helix in telomerase RNA is essential and binds to the TERT catalytic protein subunit

10.1101/2020.01.10.902601 ◽

2020 ◽

Cited By ~ 1

Author(s):

Melissa A. Mefford ◽

Evan P. Hass ◽

David C. Zappulla

Keyword(s):

Telomerase Activity ◽

Genome Replication ◽

Telomerase Rna ◽

Protein Subunit ◽

Base Pairs ◽

Binding Interaction ◽

Catalytic Core ◽

Yeast Telomerase

ABSTRACTThe telomerase RNP counters the chromosome end-replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (TERT). Although the majority of the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4-base pairs of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas ΔCEH, 1-bp, and 2-bp alleles do not support telomerase function. Using the CRISPR/dCas9-based “CARRY two-hybrid” assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nucleotides that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices strongly suggest that this binding interaction with TERT is dictated more by secondary than primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.

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Dual Role of the Alternative Reading Frame ARF Protein in Cancer

Biomolecules ◽

10.3390/biom9030087 ◽

2019 ◽

Vol 9 (3) ◽

pp. 87 ◽

Cited By ~ 11

Author(s):

Rosa Fontana ◽

Michela Ranieri ◽

Girolama La Mantia ◽

Maria Vivo

Keyword(s):

Tumor Suppressor ◽

Molecular Mechanisms ◽

Point Mutations ◽

Reading Frame ◽

Independent Functions ◽

One Year ◽

Established Role ◽

Overlapping Transcripts ◽

Median Survival Rate

The CDKN2a/ARF locus expresses two partially overlapping transcripts that encode two distinct proteins, namely p14ARF (p19Arf in mouse) and p16INK4a, which present no sequence identity. Initial data obtained in mice showed that both proteins are potent tumor suppressors. In line with a tumor-suppressive role, ARF-deficient mice develop lymphomas, sarcomas, and adenocarcinomas, with a median survival rate of one year of age. In humans, the importance of ARF inactivation in cancer is less clear whereas a more obvious role has been documented for p16INK4a. Indeed, many alterations in human tumors result in the elimination of the entire locus, while the majority of point mutations affect p16INK4a. Nevertheless, specific mutations of p14ARF have been described in different types of human cancers such as colorectal and gastric carcinomas, melanoma and glioblastoma. The activity of the tumor suppressor ARF has been shown to rely on both p53-dependent and independent functions. However, novel data collected in the last years has challenged the traditional and established role of this protein as a tumor suppressor. In particular, tumors retaining ARF expression evolve to metastatic and invasive phenotypes and in humans are associated with a poor prognosis. In this review, the recent evidence and the molecular mechanisms of a novel role played by ARF will be presented and discussed, both in pathological and physiological contexts.

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