Intracellular trafficking of yeast telomerase components

doi:10.1093/EMBO-REPORTS/KVF133

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Intracellular trafficking of yeast telomerase components

Journal Article•DOI•

Intracellular trafficking of yeast telomerase components

M.Teresa Teixeira¹, Klaus Förstemann¹, Susan M. Gasser², Joachim Lingner¹•Institutions (2)

ISREC¹, University of Geneva²

01 Jul 2002-EMBO Reports (John Wiley & Sons, Ltd)-Vol. 3, Iss: 7, pp 652-659

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.

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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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Dissertation•

Investigating the role of the n-terminus of yeast telomerase reverse transcriptase in telomere maintenance.

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Hong Ji

06 Dec 2007

Dissertation•

Dynamique intranucléaire et biogenèse des ARNs H/ACA

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Patricia Richard

01 Jan 2006

TL;DR: In this paper, the presence of a signal particulier au niveau des ARNs H/ACA guidant les modifications des snARNs and permettant leur accumulation dans les Cajal bodies.

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Abstract: Les ARNs H/ACA sont de petits ARNs nucleaires qui remplissent des fonctions variees dans la cellule. Ils servent d'ARNs guides pour les conversions d'uridines en pseudouridines des ARNs ribosomiques et des snARNs du spliceosome. Nous avons mis en evidence la presence d'un signal particulier au niveau des ARNs H/ACA guidant les modifications des snARNs et permettant leur accumulation dans les Cajal bodies. Ce signal est egalement present au niveau de l'ARN de la telomerase qui s'accumule egalement dans les Cajal bodies. Grâce a des experiences de microscopie a fluorescence, nous avons apporte des elements importants permettant d'emettre l'hypothese que l'ARN de la telomerase est delivre au niveau d'une sous-population de telomeres en phase S du cycle cellulaire. Finalement, nos travaux sur l'expression et la maturation des ARNs H/ACA introniques montrent que l'epissage et l'assemblage de la particule H/ACA chez l'Homme sont deux evenements independants.

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Dissertation•

Biogenesis of Yeast Telomerase Occurs through Two Different Nuclear Import Pathways

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Charlene Hawkins

02 Jul 2014

Architecture and Biogenesis of the Human Telomerase Holoenzyme

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Emily Denise Egan

01 Jan 2012

TL;DR: The RNA and RNP architecture of the hTR H/ACA domain is investigated and the sequence and structural elements required for RNA accumulation and R NP assembly are defined and it is found that an hTR-specific BIO box motif within the 3'H/ACA loop stimulates H/ ACA core RNP assembly in a manner required for hTR accumulation in vivo.

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Abstract: Telomerase adds simple sequence repeats to the ends of linear chromosomes to counteract the loss of end sequence inherent in conventional DNA replication. Telomerase is a ribonucleoprotein (RNP) whose catalytic activity arises from the cooperation of the telomerase reverse transcriptase protein (TERT) and the telomerase RNA (TER), which provides an integrated template for DNA repeat addition. TERs vary widely in sequence and structure but share a set of motifs required for TERT binding and catalytic activity. Species-specific TER motifs play essential roles in RNP biogenesis, stability, trafficking, and regulation. Human TER, or hTR, contains a motif shared with a large family of H/ACA RNAs that directs a pathway of RNP maturation. I have investigated the RNA and RNP architecture of the hTR H/ACA domain and defined the sequence and structural elements required for RNA accumulation and RNP assembly. I have shown that, like other eukaryotic H/ACA RNAs, hTR recruits two sets of H/ACA core proteins despite a noncanonical spacing of conserved elements in its 5' H/ACA hairpin (Chapter Two). I have found that an hTR-specific BIO box motif within the 3' H/ACA loop stimulates H/ACA core RNP assembly in a manner required for hTR accumulation in vivo (Chapter Three). Finally, I have generated stable cell lines expressing tagged hTR and TERT to enable RNA-based and protein-based affinity purification of telomerase complexes in order to identify hTR-interacting factors and investigate telomerase multimerization (Chapter Four).

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Cites background from "Intracellular trafficking of yeast ..."

...proteins bind TLC1 in the cytoplasm and facilitate its nuclear reentry as an assembled RNP (Gallardo et al., 2008; Teixeira et al., 2002)....
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...…are required for endogenous TLC1 nuclear localization and that overexpressed Est1p and Est2p localize to the nucleus suggests the possibility that these proteins bind TLC1 in the cytoplasm and facilitate its nuclear reentry as an assembled RNP (Gallardo et al., 2008; Teixeira et al., 2002)....
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Étude de la dynamique du trafic nucléo-cytoplasmique et de l’assemblage de la ribonucléoprotéine télomérase chez Saccharomyces cerevisiae

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Emmanuel Bajon

01 Jan 2017

Cites background from "Intracellular trafficking of yeast ..."

...25 25 Deux études portant sur la localisation de protéines Est fusionnées à la protéine fluorescente verte (Green Fluorescent Protein, ou GFP) et surexprimées ont montré un signal nucléaire, avec une forte composante au nucléole (Teixeira et al. 2002; Hawkins & Friedman 2014)....
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...En effet, lorsque surexprimé, Tlc1 se trouve principalement au noyau dans des levures, ce qui est attendu car la télomérase agit aux télomères, donc dans le noyau (Ferrezuelo et al. 2002; Teixeira et al. 2002)....
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...De plus, des expériences de FISH sur des cellules hétérocaryotes (i.e. possédant deux noyaux complets génétiquement différents) ont permis d’observer que Tlc1, jusqu’alors considéré comme un ARN exclusivement nucléaire, pouvait passer d’un noyau à l’autre (Teixeira et al. 2002)....
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...24 24 surexpression du gène (Gallardo et al. 2008; Teixeira et al. 2002)....
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Journal Article•DOI•

A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.

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Carol W. Greider¹, Elizabeth H. Blackburn²•Institutions (2)

Cold Spring Harbor Laboratory¹, University of California, Berkeley²

26 Jan 1989-Nature

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.

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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.

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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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Journal Article•DOI•

Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase

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Joachim Lingner¹, Timothy P. Hughes², Andrej Shevchenko², Matthias Mann², Victoria Lundblad², Thomas R. Cech² - Show less +2 more•Institutions (2)

Howard Hughes Medical Institute¹, Baylor College of Medicine²

25 Apr 1997-Science

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.

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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.

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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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Journal Article•DOI•

A telomerase component is defective in the human disease dyskeratosis congenita

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James R. Mitchell¹, Emily Wood¹, Kathleen Collins¹•Institutions (1)

University of California, Berkeley¹

02 Dec 1999-Nature

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.

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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.

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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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Journal Article•DOI•

A mutant with a defect in telomere elongation leads to senescence in yeast.

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Victoria Lundblad¹, Jack W. Szostak¹•Institutions (1)

Harvard University¹

19 May 1989-Cell

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).

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931 citations

Journal Article•DOI•

Ribosome synthesis in Saccharomyces cerevisiae.

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J. Venema¹, David Tollervey•Institutions (1)

VU University Amsterdam¹

01 Jan 1999-Annual Review of Genetics

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.

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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.

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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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