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
References
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TL;DR: It is demonstrated that CBC mediates the effect of the cap structure in U snRNA export, and direct evidence for the involvement of a cellular RNA-binding factor in the transport of RNA to the cytoplasm is provided.
Abstract: CAP structures are added cotranscriptionally to all RNA polymer-ase II transcripts1,2. They affect several processes including RNA stability3–5, pre-messenger RNA splicing6–11, RNA export from the nucleus12–14 and translation initiation15–17. The effect of the cap on translation is mediated by the initiation factor eIF-4F (refs 15–19), whereas the effect on pre-mRNA splicing involves a nuclear complex (CBC) composed of two cap binding proteins, CBP80 and CBP2011. A role for CBC in the nuclear export of capped RNAs has also been proposed11,13. We report here the characterization of human and Xenopus CBP20s. Antibodies against recombinant CBP20 prevent interaction of CBC with capped RNAs in vitro. Following microinjection into Xenopus oocytes, the antibodies inhibit both pre-mRNA splicing and export of U small nuclear RNAs to the cytoplasm. These results demonstrate that CBC mediates the effect of the cap structure in U snRNA export, and provide direct evidence for the involvement of a cellular RNA-binding factor in the transport of RNA to the cytoplasm.
341 citations
TL;DR: The efficiency of trimethylation is reduced by deleting the Sm binding site from U2 RNA, but it is not altered by other mutations in the coding sequence, and the trimethylase activity in Xenopus laevis oocytes is cytoplasmic.
325 citations
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TL;DR: An inherently high stalling frequency of the S. cerevisiae telomerase may account for its in vitro properties and for the irregular telomeric sequences of this yeast.
Abstract: The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA by copying an internal RNA template sequence. The telomerase activities of the yeasts Saccharomyces castellii and Saccharomyces cerevisiae--with regular and irregular telomeric sequences, respectively--have now been identified and characterized. The S. cerevisiae activity required the telomerase RNA gene TLC1 but not the EST1 gene, both of which are required for normal telomere maintenance in vivo. This activity exhibited low processivity and produced no regularly repeated products. An inherently high stalling frequency of the S. cerevisiae telomerase may account for its in vitro properties and for the irregular telomeric sequences of this yeast.
306 citations
"Intracellular trafficking of yeast ..." refers result in this paper
...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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TL;DR: It is shown that the Saccharomyces cerevisiae telomerase RNA has a 5′-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing.
Abstract: Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells1 and is associated with most human cancers2. In all eukaryotes examined, telomerase has an RNA subunit3, a conserved reverse transcriptase subunit4 and additional proteins5,6, but little is known about the assembly of these components. Here we show that the Saccharomyces cerevisiae telomerase RNA7 has a 5′-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing8,9. Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex. These data support a model in which telomerase RNA is transcribed by RNA polymerase II (ref. 10) and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits. We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication.
283 citations
"Intracellular trafficking of yeast ..." refers background in this paper
..., 1997), hypermethylation at the 5′ end, association with the Sm proteins (Seto et al., 1999) and association with Est proteins....
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...In Saccharomyces cerevisiae, telomerase assembly involves association of three Ever Shorter Telomeres (EST1–3) gene products and presumably seven Sm proteins with the telomerase RNA moiety TLC1 (Lundblad and Szostak, 1989; Singer and Gottschling, 1994; Lendvay et al., 1996; Seto et al., 1999; Hughes et al., 2000)....
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...The RNA subunit is subject to several maturation and assembly steps involving polyadenylation (Chapon et al., 1997), hypermethylation at the 5′ end, association with the Sm proteins (Seto et al., 1999) and association with Est proteins....
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...The TLC1 transcript has a trimethyl G cap and is associated with Sm proteins, which also bind to snRNAs essential for intron splicing (Seto et al., 1999)....
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...The TLC1 transcript has a trimethyl G cap and is associated with Sm proteins, which also bind to snRNAs essential for intron splicing (Seto et al., 1999)....
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TL;DR: This work has identified a major 38 kd nucleolar protein (NOP1), which is located within nucleolar structures resembling the dense fibrillar region of mammalian nucleoli, and suggests that NOP1 is in association with small nucleolar RNAs, required for rRNA processing and likely to be the homologue of the mammalianfibrillarin.
Abstract: In order to study the structural and functional organization of the eukaryotic nucleolus, we have started to isolate and characterize nucleolar components of the yeast Saccharomyces cerevisiae. We have identified a major 38 kd nucleolar protein (NOP1), which is located within nucleolar structures resembling the dense fibrillar region of mammalian nucleoli. This 38 kd protein is conserved in evolution since affinity-purified antibodies against the yeast protein stain the nucleolus of mammalian cells in indirect immunofluorescence microscopy and the yeast protein is decorated by antibodies directed against human fibrillarin. Affinity-purified antibodies against the yeast NOP1 efficiently precipitate at least seven small nuclear RNAs involved in rRNA maturation. We have cloned the gene encoding the yeast NOP1 protein. Haploid cells carrying a disrupted copy of the gene are not viable, showing that NOP1 is essential for cell growth. The gene codes for a 34.5 kd protein which contains glycine/arginine rich sequence repeats at the amino terminus similar to those found in other nucleolar proteins. This suggests that NOP1 is in association with small nucleolar RNAs, required for rRNA processing and likely to be the homologue of the mammalian fibrillarin.
278 citations
"Intracellular trafficking of yeast ..." refers background in this paper
...Multi- nucleated cells derived from zygotes were unambiguously identified by containing at least one nucleus that stained positive for TLC1 and GFP–Nop1p....
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...Rap1p and Nop1p immunostaining was performed as described previously (Gotta et al., Table I. Yeast strains aGasser strain collection. bObtained from B. Dujon. cFairhead et al. (1996). dThis work. eVallen et al. (1992)....
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...This zone was identified as the nucleolus by costaining with the nucleolar protein Nop1p (Schimmang et al., 1989)....
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