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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

ABSTRACT

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.

    • "In vertebrate TR, the template boundary is defined by the core-enclosing P1b helix that restricts the availability of residues to be used as templates (Chen and Greider 2003). In contrast, the template boundary in fungal TR is defined by the template-adjacent helix (Tzfati et al. 2000; Dandjinou et al. 2004; Leonardi et al. 2008; Webb and Zakian 2008). "
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    ABSTRACT: Telomerase is a ribonucleoprotein (RNP) enzyme that requires an integral telomerase RNA (TR) subunit, in addition to the catalytic telomerase reverse transcriptase (TERT), for enzymatic function. The secondary structures of TRs from the three major groups of species, ciliates, fungi, and vertebrates, have been studied extensively and demonstrate dramatic diversity. Herein, we report the first comprehensive secondary structure of TR from echinoderms-marine invertebrates closely related to vertebrates-determined by phylogenetic comparative analysis of 16 TR sequences from three separate echinoderm classes. Similar to vertebrate TR, echinoderm TR contains the highly conserved template/pseudoknot and H/ACA domains. However, echinoderm TR lacks the ancestral CR4/5 structural domain found throughout vertebrate and fungal TRs. Instead, echinoderm TR contains a distinct simple helical region, termed eCR4/5, that is functionally equivalent to the CR4/5 domain. The urchin and brittle star eCR4/5 domains bind specifically to their respective TERT proteins and stimulate telomerase activity. Distinct from vertebrate telomerase, the echinoderm TR template/pseudoknot domain with the TERT protein is sufficient to reconstitute significant telomerase activity. This gain-of-function of the echinoderm template/pseudoknot domain for conferring telomerase activity presumably facilitated the rapid structural evolution of the eCR4/5 domain throughout the echinoderm lineage. Additionally, echinoderm TR utilizes the template-adjacent P1.1 helix as a physical template boundary element to prevent nontelomeric DNA synthesis, a mechanism used by ciliate and fungal TRs. Thus, the chimeric and eccentric structural features of echinoderm TR provide unparalleled insights into the rapid evolution of telomerase RNP structure and function.
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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.
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    • "We therefore used a biochemical approach to identify the gene of interest. The experiment was based on the strategy described previously for the identification of S. pombe telomerase RNA (Leonardi et al. 2008; Webb and Zakian 2008). We used recombinant H. polymorpha TERT (Smekalova et al. 2012) to obtain antibodies for the immunoprecipitation of the telomerase complex. "
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    ABSTRACT: Telomerase, a ribonucleoprotein, is responsible for the maintenance of eukaryotic genome integrity by replicating the ends of chromosomes. The core enzyme comprises the conserved protein TERT and an RNA subunit (TER) that, in contrast, displays large variations in size and structure. Here, we report the identification of the telomerase RNA from thermotolerant yeast Hansenula polymorpha (HpTER) and describe its structural features. We show further that the H. polymorpha telomerase reverse transcribes the template beyond the predicted boundary and adds a nontelomeric dT in vitro. Sequencing of the chromosomal ends revealed that this nucleotide is specifically present as a terminal nucleotide at the 3' end of telomeres. Mutational analysis of HpTER confirmed that the incorporation of dT functions to limit telomere length in this species.
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