Intracellular trafficking of yeast telomerase components
TL;DR: It is found that Est1p, Est2p and TLC1 can migrate independently of each other to the nucleus and a role of the nucleolus in telomerase biogenesis is suggested.
Abstract: Telomerase uses an internal RNA moiety as template for the synthesis of telomere repeats. In Saccharomyces cerevisiae, the telomerase holoenzyme contains the telomerase reverse transcriptase subunit Est2p, the telomerase RNA moiety TLC1, the telomerase associated proteins Est1p and Est3p, and Sm proteins. Here we assess telomerase assembly by determining the localization of telomerase components. We found that Est1p, Est2p and TLC1 can migrate independently of each other to the nucleus. With limiting amounts of TLC1, overexpressed Est1p and Est2p accumulated in the nucleolus, whereas enzymatically active Est2p–TLC1 complexes are distributed over the entire nucleus. The distribution to the nucleoplasm depended on the specific interaction between Est2p and TLC1 but was independent of Est1p and Est3p. Altogether, our results suggest a role of the nucleolus in telomerase biogenesis. We also describe experiments that support a transient cytoplasmic localization of TLC1 RNA.
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TL;DR: It seems likely that many of the newly identified pathways/processes that affect growth of budding yeast cdc13-1 mutants will play evolutionarily conserved roles at telomeres.
Abstract: In Saccharomyces cerevisiae, Cdc13 binds telomeric DNA to recruit telomerase and to "cap" chromosome ends. In temperature-sensitive cdc13-1 mutants telomeric DNA is degraded and cell-cycle progression is inhibited. To identify novel proteins and pathways that cap telomeres, or that respond to uncapped telomeres, we combined cdc13-1 with the yeast gene deletion collection and used high-throughput spot-test assays to measure growth. We identified 369 gene deletions, in eight different phenotypic classes, that reproducibly demonstrated subtle genetic interactions with the cdc13-1 mutation. As expected, we identified DNA damage checkpoint, nonsense-mediated decay and telomerase components in our screen. However, we also identified genes affecting casein kinase II activity, cell polarity, mRNA degradation, mitochondrial function, phosphate transport, iron transport, protein degradation, and other functions. We also identified a number of genes of previously unknown function that we term RTC, for restriction of telomere capping, or MTC, for maintenance of telomere capping. It seems likely that many of the newly identified pathways/processes that affect growth of budding yeast cdc13-1 mutants will play evolutionarily conserved roles at telomeres. The high-throughput spot-testing approach that we describe is generally applicable and could aid in understanding other aspects of eukaryotic cell biology.
88 citations
TL;DR: It is shown that has1p is an essential trans‐acting factor involved in 40S ribosomal subunit biogenesis, and sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and that a has1 null strain can be fully complemented by the Candida albicans homologue.
Abstract: The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre-60S ribosomal complexes. Here, we show that Has1p is an essential trans-acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature-sensitive has1-1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre-rRNA processing by pulse-chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre-rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.
85 citations
Cites methods from "Intracellular trafficking of yeast ..."
...The resulting JDY10-3B [pHT4467-HA-HAS1] strain was then transformed with a pUN100-GFP-NOP1 plasmid (CEN, LEU2) and grown in selective medium at 30∞C to an OD600 of 0.8 (Teixeira et al., 2002)....
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TL;DR: This emerging field has uncovered that nuclear export inhibition is well poised as an attractive target towards low-toxicity broad-spectrum potent anti-cancer therapy.
Abstract: Treating cancer through inhibition of nuclear export is one of the best examples of basic research translation into clinical application. Nuclear export factor chromosomal region maintenance 1 (CRM1; Xpo1 and exportin-1) controls cellular localization and function of numerous proteins that are critical for the development of many cancer hallmarks. The diverse actions of CRM1 are likely to explain the broad ranging anti-cancer potency of CRM1 inhibitors observed in pre-clinical studies and/or clinical trials (phase I-III) on both advanced-stage solid and hematological tumors. In this review, we compare and contrast the mechanisms of action of different CRM1 inhibitors, and discuss the potential benefit of unexplored non-covalent CRM1 inhibitors. This emerging field has uncovered that nuclear export inhibition is well poised as an attractive target towards low-toxicity broad-spectrum potent anti-cancer therapy.
80 citations
TL;DR: It is proposed that yPinX1p regulates telomersase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.
Abstract: Human TRF1-binding protein PinX1 inhibits telomerase activity. Here we report that overexpression of yeast PinX1p (yPinX1p) results in shortened telomeres and decreased in vitro telomerase activity. yPinX1p coimmunoprecipitated withyeast telomerase protein Est2p even in cells lacking the telomerase RNA TLC1, or the telomerase-associated proteins Est1p and Est3p. Est2p regions required for binding to yPinX1p or TLC1 were similar. Furthermore, we found two distinct Est2p complexes exist, containing either yPinX1p or TLC1. Levels of Est2p–yPinX1p complex increased when TLC1 was deleted and decreased when TLC1 was overexpressed. Hence, we propose that yPinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.
72 citations
Cites background from "Intracellular trafficking of yeast ..."
...PinX1p, like overexpressed Est2p, is predominantly a nucleolar protein, but is also found in the nucleoplasm (Zhou and Lu 2001; Teixeira et al. 2002)....
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...cerevisiae, overexpressed Est2p accumulated in the nucleolus but was found throughout the nucleus upon TLC1 overexpression (Teixeira et al. 2002)....
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...In S. cerevisiae, overexpressed Est2p accumulated in the nucleolus but was found throughout the nucleus upon TLC1 overexpression (Teixeira et al. 2002)....
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...This redistribution of overexpressed Est2p correlated with increased in vitro telomerase activity, consistent with the interpretation that enzymatically active telomerase is in the nucleoplasm outside the nucleolus (Teixeira et al. 2002)....
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...Under what physiological situations might PinX1p act to regulate telomerase activity? PinX1p, like overexpressed Est2p, is predominantly a nucleolar protein, but is also found in the nucleoplasm (Zhou and Lu 2001; Teixeira et al. 2002)....
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TL;DR: New base‐pair formation at the 3′ end of the substrate during elongation coincides with disruption of base‐ Pair interactions at the other side of the template, Presumably, this circumvents the generation of an exceedingly high energy barrier for translocation and dissociation.
Abstract: Telomerase is the ribonucleoprotein reverse transcriptase that adds telomeric DNA repeats to the ends of chromosomes. This involves annealing of the telomerase RNA template to the 3' end of the chromosome, reverse transcription of the RNA template by the telomerase reverse transcriptase polypeptide and translocation. Here, we overexpress and partially purify the catalytically active yeast telomerase core in its natural host and probe telomerase RNA base methylation accessibility with dimethyl sulphate in the presence and absence of a DNA substrate and after substrate elongation. The length of the RNA-DNA hybrid is kept constant at seven base pairs after primer binding and elongation. Thus, new base-pair formation at the 3' end of the substrate during elongation coincides with disruption of base-pair interactions at the other side of the template. Presumably, this circumvents the generation of an exceedingly high energy barrier for translocation and dissociation. Our analysis also corroborates recently proposed yeast telomerase RNA secondary structure models.
69 citations
Cites background from "Intracellular trafficking of yeast ..."
...…pattern indicates that most of the bound substrates became elongated by five nucleotides (Fig 1C), as expected from analysis of product length obtained under similar reaction conditions (Prescott & Blackburn, 1997; Forstemann & Lingner, 2001; Teixeira et al, 2002; supplementary Fig 2 online)....
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...These two components form the catalytic core of telomerase, and their overexpression leads to increased in vitro telomerase activity without strongly affecting telomere length (Teixeira et al, 2002)....
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References
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TL;DR: The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.
Abstract: The telomerase enzyme of Tetrahymena synthesizes repeats of the telomeric DNA sequence TTGGGG de novo in the absence of added template. The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.
1,623 citations
"Intracellular trafficking of yeast ..." refers background in this paper
...Telomerase is a ribonucleoprotein (RNP) polymerase that uses an internal RNA moiety as template for the synthesis of telomere repeats (Greider and Blackburn, 1989; Lingner et al., 1997b)....
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TL;DR: The reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.
Abstract: Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.
1,293 citations
"Intracellular trafficking of yeast ..." refers background or result in this paper
...Telomerase is a ribonucleoprotein (RNP) polymerase that uses an internal RNA moiety as template for the synthesis of telomere repeats (Greider and Blackburn, 1989; Lingner et al., 1997b)....
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...Thus, Est1p and Est3p do not influence the steady-state localization of Est2p and TLC1 or their association, which is consistent with previous studies showing that Est1p and Est3p are not required for telomerase activity in vitro (Cohn and Blackburn, 1995; Lingner et al., 1997a)....
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...Active telomerase is localized in the nucleoplasm Telomerase activity requires Est2p and TLC1, which together make up the catalytic core (Lingner et al., 1997b)....
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TL;DR: It is found that primary fibroblasts and lymphoblasts from DKC-affected males are not detectably deficient in conventional H/ACA small nucleolar RNA accumulation or function; however, DKC cells have a lower level of telomerase RNA, produce lower levels of telomersase activity and have shorter telomeres than matched normal cells.
Abstract: The X-linked form of the human disease dyskeratosis congenita (DKC) is caused by mutations in the gene encoding dyskerin1. Sufferers have defects in highly regenerative tissues such as skin and bone marrow, chromosome instability and a predisposition to develop certain types of malignancy. Dyskerin is a putative pseudouridine synthase, and it has been suggested that DKC may be caused by a defect in ribosomal RNA processing. Here we show that dyskerin is associated not only with H/ACA small nucleolar RNAs2, but also with human telomerase RNA, which contains an H/ACA RNA motif3. Telomerase adds simple sequence repeats to chromosome ends using an internal region of its RNA as a template4, and is required for the indefinite proliferation of primary human cells5. We find that primary fibroblasts and lymphoblasts from DKC-affected males are not detectably deficient in conventional H/ACA small nucleolar RNA accumulation or function; however, DKC cells have a lower level of telomerase RNA, produce lower levels of telomerase activity and have shorter telomeres than matched normal cells. The pathology of DKC is consistent with compromised telomerase function leading to a defect in telomere maintenance, which may limit the proliferative capacity of human somatic cells in epithelia and blood.
1,122 citations
"Intracellular trafficking of yeast ..." refers background in this paper
...In support of this, it was shown that vertebrate telomerase RNA contains an H/ACA motif that targets the RNA to nucleoli where it was hypothesized to associate with the catalytic subunit of telomerase (Mitchell et al., 1999; Lukowiak et al., 2001)....
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TL;DR: Using this assay, a mutant that displays a progressive decrease in telomere length as well as an increased frequency of chromosome loss is isolated, which defines a new gene, designated EST1 (for ever shorter telomeres).
931 citations
TL;DR: The recent, and often surprising, advances in the understanding of ribosome synthesis in the yeast Saccharomyces cerevisiae will underscore the unexpected complexity of eukaryotic ribosomes synthesis.
Abstract: The synthesis of ribosomes is one of the major metabolic pathways in all cells. In addition to around 75 individual ribosomal proteins and 4 ribosomal RNAs, synthesis of a functional eukaryotic ribosome requires a remarkable number of trans-acting factors. Here, we will discuss the recent, and often surprising, advances in our understanding of ribosome synthesis in the yeast Saccharomyces cerevisiae. These will underscore the unexpected complexity of eukaryotic ribosome synthesis.
779 citations
"Intracellular trafficking of yeast ..." refers background in this paper
...Since the nucleolus is considered to be a site with a high concentration of trans-acting factors required for the assembly of ribosomes and other RNPs (Pederson, 1998; Sleeman and Lamond, 1999; Venema and Tollervey, 1999), it may provide factors that assist in the assembly of telomerase....
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