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 is reported that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres.
Abstract: A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3′end of the mature RNA molecule. Here, we report that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.
50 citations
Cites background from "Intracellular trafficking of yeast ..."
...It has been well documented that the telomerase travels to the cytoplasm, however the biological significance of the cytoplasmic phase on the regulation of telomerase RNA remains unclear [32,33,34]....
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...to shuttle through the cytoplasm in yeast and mammalian cells [32,33,34]....
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TL;DR: How tracking the timing of their dance during the cell cycle will yield insights into chromosome stability mechanisms is summarized, which is of particular interest for the understanding of cancer initiation or progression.
42 citations
TL;DR: Flexible Sm rings associate with and dissociate from RNA substrates more freely than fixed rings, which suggests that the conformation of flexible Sm rings might be modified in some specific manner to facilitate association and dissociation with RNA.
Abstract: The Sm family of proteins is closely associated with RNA metabolism throughout all life. These proteins form homomorphic and heteromorphic rings consisting of six or seven subunits with a characteristic central pore, the presence of which is critical for binding U-rich regions of single-stranded RNA. Eubacteria and Archaea typically carry one or two forms of Sm proteins and assemble one homomorphic ring per Sm protein. Eukaryotes typically carry 16 or more Sm proteins that assemble to form heteromorphic rings which lie at the center of a number of critical RNA-associated small nuclear ribonucleoproteins (snRNPs). High Sm protein diversity and heteromorphic Sm rings are features stretching back to the origin of eukaryotes; very deep phylogenetic divisions among existing Sm proteins indicate simultaneous evolution across essentially all existing eukaryotic life. Two basic forms of heteromorphic Sm rings are found in eukaryotes. Fixed Sm rings are highly stable and static and are assembled around an RNA cofactor. Flexible Sm rings also stabilize and chaperone RNA but assemble in the absence of an RNA substrate and, more significantly, associate with and dissociate from RNA substrates more freely than fixed rings. This suggests that the conformation of flexible Sm rings might be modified in some specific manner to facilitate association and dissociation with RNA. Diversification of eukaryotic Sm proteins may have been initiated by gene transfers and/or genome clashes that accompanied the origin of the eukaryotic cell itself, with further diversification driven by a greater need for steric specificity within increasingly complex snRNPs.
41 citations
Cites background from "Intracellular trafficking of yeast ..."
...Yeast telomerase snRNPs have been shown to be assembled in the cytoplasm and imported into the nucleus (Ferrezuelo et al. 2002; Teixeira et al. 2002), and in humans, telomerase components are found in Cajal bodies (Fu and Collins 2006), suggesting that maturation pathways in at least somemetazoans…...
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...The analyses presented here provide insights into the evolution of this distinctive, ubiquitous, and critical family of RNA-associated proteins....
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TL;DR: It is shown that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation and the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomersase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells.
Abstract: In addition to performing its canonical function, Telomerase Reverse Transcriptase (TERT) has been shown to participate in cellular processes independent of telomerase activity. Furthermore, although TERT mainly localizes to Cajal bodies, it is also present within the nucleolus. Because the nucleolus is the site of rDNA transcription, we investigated the possible role of telomerase in regulating RNA polymerase I (Pol I). Here we show that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation. Moreover, the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomerase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells. In vitro, telomerase can stimulate the formation of the transcription initiation complex. Our results demonstrate how non-canonical features of telomerase may direct Pol I transcription in oncogenic and regenerative hyperproliferation.
35 citations
TL;DR: It is suggested that the formation of GFCs is associated with a high rate of ribosome biogenesis of the transcriptionally more active large-size neurons.
34 citations
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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