Webb, C.J. & Zakian, V.A. Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA. Nat. Struct. Mol. Biol. 15, 34-42

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 02/2008; 15(1):34-42. DOI: 10.1038/nsmb1354
Source: PubMed


Although the catalytic subunit of the Schizosaccharomyces pombe telomerase holoenzyme was identified over ten years ago, the unusual heterogeneity of its telomeric DNA made it difficult to identify its RNA component. We used a new two-step immunoprecipitation and reverse transcription-PCR technique to identify the S. pombe telomerase RNA, which we call TER1. TER1 RNA was 1,213 nucleotides long, similar in size to the Saccharomyces cerevisiae telomerase RNA, TLC1. TER1 RNA associated in vivo with the two known subunits of the S. pombe telomerase holoenzyme, Est1p and Trt1p, and neither association was dependent on the other holoenzyme component. We present a model to explain how telomerase introduces heterogeneity into S. pombe telomeres. The technique used here to identify TER1 should be generally applicable to other model organisms.

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    • "We hypothesize that the conserved core elements proposed previously ( Lin et al , 2004 ) are maintained in a particular order in telo - merase to maintain the ARC and that the ARC represents an overarching conserved feature of nearly all telomerase RNAs . Examination of 107 available TER secondary structure models from ciliate , vertebrate , and fungal species ( Lingner et al , 1994 ; McCormick - Graham and Romero , 1995 ; Chen et al , 2000 ; Dandjinou et al , 2004 ; Zappulla and Cech , 2004 ; Brown et al , 2007 ; Podlevsky et al , 2008 ; Webb and Zakian , 2008 ; Xie et al , 2008 ; Qi et al , 2012 ; Li et al , 2013 ) shows that 97% of telomerase RNAs have an intact ARC , as defined by having the pseudoknot connected to the template region via a CEH ( Figure 9 ) . As for the 3% ( three species ) without an intact ARC , they are a subset of the rodent lineage , which have their 5 0 end just upstream of the template and thus lack a CEH . "
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    ABSTRACT: Telomerase is a specialized chromosome end-replicating enzyme required for genome duplication in many eukaryotes. An RNA and reverse transcriptase protein subunit comprise its enzymatic core. Telomerase is evolving rapidly, particularly its RNA component. Nevertheless, nearly all telomerase RNAs, including those of H. sapiens and S. cerevisiae, share four conserved structural elements: a core-enclosing helix (CEH), template-boundary element, template, and pseudoknot, in this order along the RNA. It is not clear how these elements coordinate telomerase activity. We find that although rearranging the order of the four conserved elements in the yeast telomerase RNA subunit, TLC1, disrupts activity, the RNA ends can be moved between the template and pseudoknot in vitro and in vivo. However, the ends disrupt activity when inserted between the other structured elements, defining an Area of Required Connectivity (ARC). Within the ARC, we find that only the junction nucleotides between the pseudoknot and CEH are essential. Integrating all of our findings provides a basic map of functional connections in the core of the yeast telomerase RNP and a framework to understand conserved element coordination in telomerase mechanism.
    The EMBO Journal 10/2013; DOI:10.1038/emboj.2013.227 · 10.43 Impact Factor
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    • "The first TER was discovered in the ciliated protozoan, Tetrahymena thermophila [7]. Subsequently, TERs have been identified from other ciliates [8], [9], [10], [11], [12], [13], vertebrates [14], yeasts [15], [16], [17], [18], [19], plants [20], and very recently in filamentous fungi [21]. Since some filamentous fungi, such as the Aspergilli, possess extremely short and tightly regulated telomeres [22], [23], they provide a unique model system to study telomere dynamics. "
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    ABSTRACT: Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5' splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.
    PLoS ONE 03/2013; 8(3):e58661. DOI:10.1371/journal.pone.0058661 · 3.23 Impact Factor
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    • "Despite the conservation of structural elements, TER is extremely divergent at the nucleotide level, making bioinformatics approaches based on sequence similarity of the TER locus difficult, even among closely related species. Notably, TER from fission yeast was uncovered only 4 years ago using a biochemical approach (Leonardi et al., 2008; Webb and Zakian, 2008), while the Caenorhabditis elegans TER has yet to be discovered. TER from the flowering plant Arabidopsis thaliana was reported in 2011, following biochemical purification of the plant enzyme (Cifuentes-Rojas et al., 2011). "
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    ABSTRACT: The telomerase reverse transcriptase promotes genome integrity by continually synthesizing a short telomere repeat sequence on chromosome ends. Telomerase is a ribonucleoprotein complex whose integral RNA subunit TER contains a template domain with a sequence complementary to the telomere repeat that is reiteratively copied by the catalytic subunit. Although TER harbors well-conserved secondary structure elements, its nucleotide sequence is highly divergent, even among closely related organisms. Thus, it has been extremely challenging to identify TER orthologs by bioinformatics strategies. Recently, TER was reported in the flowering plant, Arabidopsis thaliana. In contrast to other model organisms, A. thaliana encodes two TER subunits, only one of which is required to maintain telomere tracts in vivo. Here we investigate the evolution of the loci that encode TER in Arabidopsis by comparison to the same locus in its close relatives. We employ a combination of PCR and bioinformatics approaches to identify putative TER loci based on syntenic regions flanking the TER1 and TER2 loci of A. thaliana. Unexpectedly, we discovered that the genomic regions encoding the two A. thaliana TERs occur as a single locus in other Brassicaceae. Moreover, we find striking sequence divergence within the telomere template domain of putative TERs from Brassicaceae, including some orthologous loci that completely lack a template domain. Finally, evolution of the locus is characterized by lineage-specific events rather than changes shared among closely related species. We conclude that the Arabidopsis TER duplication occurred very recently, and further that changes at this locus in other Brassicaceae indicate the process of TER evolution may be different in plants compared with vertebrates and yeast.
    Frontiers in Genetics 09/2012; 3:188. DOI:10.3389/fgene.2012.00188
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