Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human

Telomerase is an RNA–protein complex that includes a unique reverse transcriptase that catalyzes the addition of single-stranded telomere DNA repeats onto the 3′ ends of linear chromosomes using an integral telomerase RNA (TR) template. Vertebrate TR contains the template/pseudoknot (t/PK) and CR4/5 domains required for telomerase activity in vitro. All vertebrate pseudoknots include two subdomains: P2ab (helices P2a and P2b with a 5/6-nt internal loop) and the minimal pseudoknot (P2b–P3 and associated loops). A helical extension of P2a, P2a.1, is specific to mammalian TR. Using NMR, we investigated the structures of the full-length TR pseudoknot and isolated subdomains in Oryzias latipes (Japanese medaka fish), which has the smallest vertebrate TR identified to date. We determined the solution NMR structure and studied the dynamics of medaka P2ab, and identified all base pairs and tertiary interactions in the minimal pseudoknot. Despite differences in length and sequence, the structure of medaka P2ab is more similar to human P2ab than predicted, and the medaka minimal pseudoknot has the same tertiary interactions as the human pseudoknot. Significantly, although P2a.1 is not predicted to form in teleost fish, we find that it forms in the full-length pseudoknot via an unexpected hairpin. Model structures of the subdomains are combined to generate a model of t/PK. These results provide evidence that the architecture for the vertebrate t/PK is conserved from teleost fish to human. The organization of the t/PK on telomerase reverse transcriptase for medaka and human is modeled based on the cryoEM structure of Tetrahymena telomerase, providing insight into function.

Download Full-text

Peculiarities of Yeasts and Human Telomerase RNAs Processing

Acta Naturae ◽

10.32607/20758251-2016-8-4-14-22 ◽

2016 ◽

Vol 8 (4) ◽

pp. 14-22 ◽

Cited By ~ 6

Author(s):

M. P. Rubtsova ◽

D. P. Vasilkova ◽

Yu. V. Naraykina ◽

O. A. Dontsova

Keyword(s):

Reverse Transcriptase ◽

Somatic Cells ◽

Telomerase Activity ◽

Proliferative Potential ◽

Telomerase Rna ◽

Embryonic Cells ◽

Eukaryotic Chromosome ◽

Human Telomerase ◽

Linear Chromosomes

Telomerase is one of the major components of the telomeres -- linear eukaryotic chromosome ends - maintenance system. Linear chromosomes are shortened during each cell division due to the removal of the primer used for DNA replication. Special repeated telomere sequences at the very ends of linear chromosomes prevent the deletion of genome information caused by primer removal. Telomeres are shortened at each replication round until it becomes critically short and is no longer able to protect the chromosome in somatic cells. At this stage, a cell undergoes a crisis and usually dies. Rare cases result in telomerase activation, and the cell gains unlimited proliferative capacity. Special types of cells, such as stem, germ, embryonic cells and cells from tissues with a high proliferative potential, maintain their telomerase activity indefinitely. The telomerase is inactive in the majority of somatic cells. Telomerase activity in vitro requires two key components: telomerase reverse transcriptase and telomerase RNA. In cancer cells, telomerase reactivates due to the expression of the reverse transcriptase gene. Telomerase RNA expresses constitutively in the majority of human cells. This fact suggests that there are alternative functions to telomerase RNA that are unknown at the moment. In this manuscript, we review the biogenesis of yeasts and human telomerase RNAs thanks to breakthroughs achieved in research on telomerase RNA processing by different yeasts species and humans in the last several years.

Download Full-text

Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions

Molecular and Cellular Biology ◽

10.1128/mcb.21.5.1888-1897.2001 ◽

2001 ◽

Vol 21 (5) ◽

pp. 1888-1897 ◽

Cited By ~ 78

Author(s):

François Bachand ◽

Chantal Autexier

Keyword(s):

Reverse Transcriptase ◽

Rna Binding ◽

Telomerase Activity ◽

Catalytic Subunit ◽

Telomerase Rna ◽

Dna Repeats ◽

Human Telomerase Reverse Transcriptase ◽

Template Sequence ◽

Human Telomerase

ABSTRACT Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.

Download Full-text

A 4-base pair core-enclosing helix in telomerase RNA is essential and binds to the TERT catalytic protein subunit

10.1101/2020.01.10.902601 ◽

2020 ◽

Cited By ~ 1

Author(s):

Melissa A. Mefford ◽

Evan P. Hass ◽

David C. Zappulla

Keyword(s):

Telomerase Activity ◽

Genome Replication ◽

Telomerase Rna ◽

Protein Subunit ◽

Base Pairs ◽

Binding Interaction ◽

Catalytic Core ◽

Yeast Telomerase

ABSTRACTThe telomerase RNP counters the chromosome end-replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (TERT). Although the majority of the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4-base pairs of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas ΔCEH, 1-bp, and 2-bp alleles do not support telomerase function. Using the CRISPR/dCas9-based “CARRY two-hybrid” assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nucleotides that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices strongly suggest that this binding interaction with TERT is dictated more by secondary than primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.

Download Full-text

Plasmodium falciparum Telomerase: De Novo Telomere Addition to Telomeric and Nontelomeric Sequences and Role in Chromosome Healing

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.919 ◽

1998 ◽

Vol 18 (2) ◽

pp. 919-925 ◽

Cited By ~ 50

Author(s):

Emmanuel Bottius ◽

Nassera Bakhsis ◽

Artur Scherf

Keyword(s):

Plasmodium Falciparum ◽

Reverse Transcriptase ◽

De Novo ◽

Telomerase Activity ◽

Telomeric Dna ◽

Oligonucleotide Primers ◽

Cell Extracts ◽

Linear Chromosomes ◽

Dna Primers

ABSTRACT Telomerase, a specialized cellular reverse transcriptase, compensates for chromosome shortening during the proliferation of most eucaryotic cells and contributes to cellular immortalization. The mechanism used by the single-celled protozoan malaria parasitePlasmodium falciparum to complete the replication of its linear chromosomes is currently unknown. In this study, telomerase activity has for the first time been identified in cell extracts ofP. falciparum. The de novo synthesis of highly variable telomere repeats to the 3′ end of DNA oligonucleotide primers by plasmodial telomerase is demonstrated. Permutated telomeric DNA primers are extended by the addition of the next correct base. In addition to elongating preexisting telomere sequences, P. falciparumtelomerase can also add telomere repeats onto nontelomeric 3′ ends. The sequence GGGTT… was the predominant initial DNA sequence added to the nontelomeric 3′ ends in vitro. Poly(C) at the 3′ end of the oligonucleotide significantly alters the precision of the new telomerase added repeats. The efficiency of nontelomeric primer elongation was dependent on the presence of a G-rich cassette upstream of the 3′ terminus. Oligonucleotide primers based on natural P. falciparum chromosome breakpoints are efficiently used as telomerase substrates. These results imply that P. falciparum telomerase contributes to chromosome maintenance and to de novo telomere formation on broken chromosomes. Reverse transcriptase inhibitors such as dideoxy GTP efficiently inhibitP. falciparum telomerase activity in vitro. These data point to malaria telomerase as a new target for the development of drugs that could induce parasite cell senescence.

Download Full-text

Functional Multimerization of the Human Telomerase Reverse Transcriptase

Molecular and Cellular Biology ◽

10.1128/mcb.21.18.6151-6160.2001 ◽

2001 ◽

Vol 21 (18) ◽

pp. 6151-6160 ◽

Cited By ~ 92

Author(s):

Tara L. Beattie ◽

Wen Zhou ◽

Murray O. Robinson ◽

Lea Harrington

Keyword(s):

Reverse Transcriptase ◽

Telomerase Activity ◽

Telomerase Reverse Transcriptase ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Catalytic Core ◽

Cell Extracts ◽

Human Telomerase

ABSTRACT The telomerase enzyme exists as a large complex (∼1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790–2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.

Download Full-text

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5131-5144.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5131-5144

Author(s):

H Wang ◽

J C Kennell ◽

M T Kuiper ◽

J R Sabourin ◽

R Saldanha ◽

...

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcription ◽

Full Length ◽

Minus Strand ◽

Ribonucleoprotein Particle ◽

Cdna Synthesis ◽

Reverse Transcriptases ◽

In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

Download Full-text

Telomerase Activity Reconstitutedin Vitrowith Purified Human Telomerase Reverse Transcriptase and Human Telomerase RNA Component

Journal of Biological Chemistry ◽

10.1074/jbc.m000622200 ◽

2000 ◽

Vol 275 (29) ◽

pp. 22568-22573 ◽

Cited By ~ 47

Author(s):

Kenkichi Masutomi ◽

Shuichi Kaneko ◽

Naoyuki Hayashi ◽

Tatsuya Yamashita ◽

Yukihiro Shirota ◽

...

Keyword(s):

Reverse Transcriptase ◽

Telomerase Activity ◽

Telomerase Reverse Transcriptase ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Human Telomerase

Download Full-text

Detection of telomerase activity, human telomerase RNA, and telomerase reverse transcriptase in human hepatocellular carcinoma

Gastroenterology ◽

10.1016/s0016-5085(00)86121-1 ◽

2000 ◽

Vol 118 (4) ◽

pp. A993

Author(s):

Masaki Shimojima ◽

Fumihiko Komine ◽

Hiroshi Aoki ◽

Toshihiro Shimizu ◽

Tamiko Kaneko ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Reverse Transcriptase ◽

Telomerase Activity ◽

Human Hepatocellular Carcinoma ◽

Telomerase Reverse Transcriptase ◽

Telomerase Rna ◽

Human Telomerase

Download Full-text

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915312116 ◽

2019 ◽

Vol 116 (49) ◽

pp. 24542-24550 ◽

Cited By ~ 8

Author(s):

Jiarui Song ◽

Dhenugen Logeswaran ◽

Claudia Castillo-González ◽

Yang Li ◽

Sreyashree Bose ◽

...

Keyword(s):

Telomerase Activity ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Telomeric Dna ◽

Structural Element ◽

Stem Loop ◽

Evolutionary Pathway ◽

Pol Iii

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

Download Full-text