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: The details of telomerase and its regulation by the telomere are discussed, including single-stranded DNA-binding proteins (POT1 in humans and Cdc13 in budding yeast), which have been proposed to contribute to the recruitment of telomersase and may also regulate the extent or frequency of elongation.
Abstract: ▪ Abstract Telomeres are essential for genome stability in all eukaryotes. Changes in telomere functions and the associated chromosomal abnormalities have been implicated in human aging and cancer. Telomeres are composed of repetitive sequences that can be maintained by telomerase, a complex containing a reverse transcriptase (hTERT in humans and Est2 in budding yeast), a template RNA (hTERC in humans and Tlc1 in yeast), and accessory factors (the Est1 proteins and dyskerin in humans and Est1, Est3, and Sm proteins in budding yeast). Telomerase is regulated in cis by proteins that bind to telomeric DNA. This regulation can take place at the telomere terminus, involving single-stranded DNA-binding proteins (POT1 in humans and Cdc13 in budding yeast), which have been proposed to contribute to the recruitment of telomerase and may also regulate the extent or frequency of elongation. In addition, proteins that bind along the length of the telomere (TRF1/TIN2/tankyrase in humans and Rap1/Rif1/Rif2 in budding y...
823 citations
TL;DR: It is demonstrated that telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states by taking a molecular snapshot of a single round of telomeres replication.
530 citations
TL;DR: Since the discovery of an enzyme that extended the DNA at chromosome telomeres in the ciliate, Tetrahymena, there has been an explosion of knowledge about both the RNA and protein subunits of this unusual ribonucleoprotein enzyme.
430 citations
Cites background from "Intracellular trafficking of yeast ..."
...Whether this functions (Ferrezuelo et al., 2002; Teixeira et al., 2002). template-recognition element directly binds to TERT or Human telomerase RNA, also a pol II transcript, has a interacts with another portion of the RNA remains a snoRNP (small nucleolar RNP) domain, appears to be question for…...
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TL;DR: Concomitant overexpression of TERT and TR was necessary and sufficient to substantially increase telomerase activity, and in less than 50 PDs, the length of telomeres increased 3–8‐fold beyond physiological size, while telomere‐bound TRF1 and TRF2 increased proportionally to telomer length.
Abstract: Stabilization of telomere length in germline and highly proliferative human cells is required for long-term survival and for the immortal phenotype of cancer-derived cells. This is achieved through expression of telomerase reverse transcriptase (TERT), which synthesizes telomeric repeats through reverse transcription of its tightly associated RNA template (TR). The telomeric repeat binding factor TRF1 inhibits telomerase at telomeres in cis in a length-dependent manner to achieve telomere length homeostasis. Here we manipulate telomerase activity over a wide range in cancer and primary cells. Concomitant overexpression of TERT and TR was necessary and sufficient to substantially increase telomerase activity. Upon overexpression, more telomerase associated with telomeres and telomeres elongated at a constant rate (up to 0.8 kb/population doubling (PD)) in a length-independent manner. Thus, in less than 50 PDs, the length of telomeres increased 3-8-fold beyond physiological size, while telomere-bound TRF1 and TRF2 increased proportionally to telomere length. Thus, long telomeres do not permanently adopt a structural state that is non-extendible. A low cellular concentration of telomerase is critical to achieve preferential elongation of short telomeres and telomere length homeostasis.
296 citations
Cites background from "Intracellular trafficking of yeast ..."
...High levels of telomerase activity result in continuous telomere elongation In budding yeast, overexpression of the TR subunit TLC1 together with the TERT subunit Est2p also results in higher telomerase activity levels in cell extracts (Teixeira et al, 2002)....
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TL;DR: Insights that have been gained into the cellular pathways for biogenesis and regulation of telomerase ribonucleoproteins raise new questions, particularly concerning the dynamic nature of this unique polymerase.
Abstract: Chromosome stability requires a dynamic balance of DNA loss and gain in each terminal tract of telomeric repeats. Repeat addition by a specialized reverse transcriptase, telomerase, has an important role in maintaining this equilibrium. Insights that have been gained into the cellular pathways for biogenesis and regulation of telomerase ribonucleoproteins raise new questions, particularly concerning the dynamic nature of this unique polymerase.
276 citations
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TL;DR: It is demonstrated that the production of active recombinant telomerase requires a factor in rabbit reticulocyte lysate that promotes ribonucleoprotein assembly and indicates that recombinant Telomerase activity requires more than the catalytic protein and RNA components of the enzyme that have been identified to date.
Abstract: Telomerase is a ribonucleoprotein reverse transcriptase specialized for use of a sequence within its integral RNA component as the template for DNA synthesis. Telomerase adds telomeric simple sequence repeats to single-stranded primers in vitro or chromosome ends in vivo. We have investigated the sequences and structures of recombinant Tetrahymena thermophila telomerase RNA necessary for physical association and activity with the catalytic protein subunit expressed in rabbit reticulocyte lysate. In contrast with previous results using another reconstitution method, we find that phylogenetically conserved primary sequences and a phylogenetically nonconserved secondary structure are essential for telomerase RNA function. Telomerase RNA binding to the catalytic protein subunit requires sequences 5' of the template and is highly sequence specific. Other telomerase RNA sequences are required for enzyme activity and proper template use but not for protein interaction affinity. In addition, we demonstrate that the production of active recombinant telomerase requires a factor in rabbit reticulocyte lysate that promotes ribonucleoprotein assembly. These studies demonstrate multiple functions for the telomerase RNA and indicate that recombinant telomerase activity requires more than the catalytic protein and RNA components of the enzyme that have been identified to date.
77 citations
"Intracellular trafficking of yeast ..." refers background in this paper
...The assembly of telomerases from other species is a chaperone-assisted process (Holt et al., 1999; Licht and Collins, 1999)....
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TL;DR: It is demonstrated that U1 snRNA is exported like an mRNA on insertion of a pre-mRNA intron or either sense or antisense mRNA exon sequences, suggesting that an unstructured region of sufficient length in an RNA acts as a dominant determinant of mRNA identity.
75 citations
"Intracellular trafficking of yeast ..." refers background in this paper
...However, we cannot rule out the possibility that the ORF itself recruited the mRNA export machinery (Ohno et al., 2002) or that translation occurred in the nucleus, although to date translation in the nucleus has only been demonstrated for very small peptides (Iborra et al., 2001)....
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...However, we cannot rule out the possibility that the ORF itself recruited the mRNA export machinery (Ohno et al., 2002) or that translation occurred in the nucleus, although to date translation in the nucleus has only been demonstrated for very small peptides (Iborra et al....
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TL;DR: The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.
Abstract: Telomerase is a ribonucleoprotein enzyme that adds repetitive sequences to the ends of linear chromosomes, thereby counteracting nucleotide loss due to incomplete replication. A short region of the telomerase RNA subunit serves as template for nucleotide addition onto the telomere 3' end. Although Saccharomyces cerevisiae contains only one telomerase RNA gene, telomere repeat sequences are degenerate in this organism. Based on a detailed analysis of the telomere sequences specified by wild-type and mutant RNA templates in vivo, we show that the divergence of telomere repeats is due to abortive reverse transcription in the 3' and 5' regions of the template and due to the alignment of telomeres in multiple registers within the RNA template. Through the interpretation of wild-type telomere sequences, we identify nucleotides in the template that are not accessible for base pairing during substrate annealing. Rather, these positions become available as templates for reverse transcription only after alignment with adjacent nucleotides has occurred, indicating that a conformational change takes place upon substrate binding. We also infer that the central part of the template region is reverse transcribed processively. The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.
74 citations
"Intracellular trafficking of yeast ..." refers methods in this paper
...(C) Telomerase assays performed as described (Forstemann and Lingner, 2001) using extracts from FYBL1-4D (lanes 1 and 2) and YKF15 (lanes 3 and 4) transformed either with pRS314 (lanes 1 and 3) or pRS314-PGAL1-TLC1 (lanes 2 and 4)....
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TL;DR: The FISH protocol to analyze the subnuclear localization of several proteins at once and to localize proteins relative to DNA sequences is described to provide a powerful tool for the characterization of nuclear protein localization and the positioning of specific chromosomal regions.
Abstract: Publisher Summary This chapter presents the analysis of nuclear organization in Saccharomyces cerevisiae and describes combined immunofluorescence fluorescent in situ hybridization (FISH) protocol to analyze the subnuclear localization of several proteins at once and to localize proteins relative to DNA sequences. Protocols presented in the chapter omit protease and harsh-detergent treatment. Cells are fixed after spheroplasting in osmotically buffered growth medium to efficiently preserve nuclear structure. In this way, the cell wall does not itself become a target of cross-linking reagents, a phenomenon that can prevent efficient diffusion of the fixative to the nucleus. At the same time, high efficiency labeling is obtained with both antibodies and DNA probes. The protocol can also be performed on cells that are fixed prior to spheroplasting if preservation of the cell shape is desired. The double in situ /immunofluorescence staining described in the chapter provides a powerful tool for the characterization of nuclear protein localization and the positioning of specific chromosomal regions.
31 citations
"Intracellular trafficking of yeast ..." refers methods in this paper
...Slides were treated for in situ hybridization as described in Gotta et al. (1999) without the RNase treatment....
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