Telomerase RNA Moiety Research Articles

The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5′-UUAGGG-3′ repeats near their 3′-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5′-UUAGGG-3′ repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

The 5′ Arm of Kluyveromyces lactis Telomerase RNA Is Critical for Telomerase Function

Telomerase is a ribonucleoprotein reverse transcriptase that copies a short template within its integral telomerase RNA moiety (TER) onto eukaryotic chromosome ends, thus compensating for incomplete replication and degradation. The highly divergent yeast TER is structured in three long arms, with a catalytic core at its center. A binding site for the protein Ku80 is conserved within the 5' arm of TER in Saccharomyces but not in Kluyveromyces budding yeast species. Consistently, KU80 deletion in Kluyveromyces lactis does not affect telomere length, while it causes telomere shortening in Saccharomyces cerevisiae. We found elements in the 5' arm of K. lactis TER that are crucial for telomerase activity and stability. However, we found no indication of the association of Ku80 with this arm. Although the overexpression of Ku80 rescues a particular mutation in K. lactis TER1 that phenocopies a telomerase null mutation, this effect is indirect, caused by the repression of the recombination pathway competing for telomere maintenance. Interestingly, the overexpression of Est3, an essential telomerase protein whose function is still unknown, suppresses the phenotypes of mutations in this arm. These results indicate that the 5' arm of K. lactis TER has critical roles in telomerase function, which may be linked to the function of Est3.

Protein–RNA and protein–protein interactions mediate association of human EST1A/SMG6 with telomerase

The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein–RNA and protein–protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein–protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall Kd = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR–hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein–protein contacts with hTERT.

Telomerase and cell senescence: modulation by telomerase of cellular responses to damage in normal human primary skin keratinocytes and fibroblasts

Telomeres protect the ends of chromosomes, which contain the DNA that comprises the genetic information. Without telomeres and their special way of replicating, chromosome ends dwindle away and also are easily damaged. Loss of telomere function promotes cancer and may occur in human aging. Most enzymes – the catalysts that carry out life's chemical reactions – are made of protein. In contrast, the enzyme telomerase, which replenishes the DNA at telomeres and protects them, is a ribonucleoprotein complex consisting of core components – a protein reverse transcriptase and the telomerase RNA moiety – as well as additional protein factors that regulate the action of telomerase on telomeres.A major known function of telomerase is to elongate telomeric DNA. This function is achieved by the enzyme copying a short template sequence within the inbuilt telomerase RNA, into telomeric DNA repeat sequences. The mechanism of telomerase has been analyzed and telomerase has been shown to be a dimeric enzyme. Functional dimerization of human telomerase requires a novel RNA–RNA interaction between two telomerase RNA molecules in the same enzyme complex. Telomeric repeats are added to the end of the chromosomal DNA by telomerase, elongating the chromosome and thus compensating for losses of terminal DNA that occur upon nuclease action and incomplete DNA replication of the ends of the chromosomal DNA.However, telomerase is largely repressed in proliferating cultured human fibroblasts or after a few passages of culturing of primary human keratinocytes. The normal senescence of such fibroblasts and other human cell types in culture can be overcome by forced expression of telomerase in these cells. The lifespan of the cells is extended and they grow healthily, generally without evidence of impairment to DNA checkpoint pathways.We analyzed the effects of forced expression of telomerase on the growth characteristics and sensitivity to ultraviolet (UV) irradiation of primary human cells in culture. Normal primary human foreskin epithelial keratinocytes and fibroblasts were stably infected with retroviral vector expressing various versions of hTERT (the reverse transcriptase protein component of telomerase), or the control empty retroviral vector pBABEpuro. Cells selected for the puromycin drug resistance marker were continuously passaged in puromycin‐containing media, and their telomeres, cell growth and cell viability analyzed. The responses of such cells expressing telomerase to different challenges, including UV exposure were analyzed and found to differ from those of control cells.

Intracellular trafficking of yeast telomerase components.

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.

Chapter 20 Studying the Telomerase RNA in Tetrahymena

Publisher Summary Telomeres are specialized structures at the ends of eukaryotic chromosomes that are required for chromosome stability and the complete replication of linear chromosomes. In ciliates de novo telomere formation is an essential step in a critical developmental stage—the generation of the new macronucleus. Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the synthesis of telomeric repeats. Telomerase is the only known reverse transcriptase that contains its own template, which is present in the telomerase RNA moiety as an integral part of the RNP complex. The telomerase RNA of Tetrahymena thermophila is a relatively small RNA (159 nucleotides) with a very well-defined secondary structure. Specific residues of the templating domain are copied to produce the G-rich telomeric strand. Ciliates devote significant cellular resources to the maintenance and replication of telomeres due to the large number of macronuclear chromosomes and the relatively small size of each macronuclear chromosome. Consequently, each cell contains an unusually large number of chromosome ends.

Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase

Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P. tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P. caudatum telomerase RNA can participate in telomere synthesis when expressed in the P. tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P. tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P. tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P. caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.

A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains.

The ribonucleoprotein (RNP) enzyme telomerase is required for replication of eukaryotic chromosomal termini. The RNA moiety of telomerase is essential for enzyme function and provides the template for telomeric DNA synthesis. However, the roles of its nontemplate domains have not been explored. Here we demonstrate that a novel interspecies telomerase RNA swap in vivo creates a functional but aberrant telomerase. Telomerase RNA from the ciliate Glaucoma chattoni was expressed in Tetrahymena thermophila cells. The telomerase RNAs from these two species have almost superimposable secondary structures. The template region base sequence is identical in the two RNAs, but elsewhere their sequences differ by 49%. This hybrid telomerase RNP was enzymatically active but added only short stretches of telomeric repeat tracts in vivo and in vitro. This new enzyme also had a strong, aberrant DNA cleavage activity in vitro. Thus, molecular interactions in the RNP involving nontemplate RNA domains affect specific aspects of telomerase enzyme function, raising the possibility that they may regulate telomerase activity.

Architecture of telomerase RNA.

Telomerase, an essential ribonucleoprotein reverse transcriptase, adds telomeric DNA to the ends of eukaryotic chromosomes. We examined the conformational properties of the naked RNA moiety of telomerase from two related ciliates, Tetrahymena thermophila and Glaucoma chattoni. As well as finding evidence for features proposed previously on the basis of phylogenetic comparisons, novel conserved structural properties were revealed. Specifically, although the region around helix III was previously proposed to form a pseudoknot, our results indicate that in the naked RNA this region maintains a level of 'plasticity', probably in an equilibrium favoring one of two helices. In addition, these studies reveal that the templating domain is not entirely single-stranded as previously proposed, but is ordered due to constraints imposed by other parts of the RNA. Finally, our results suggest that the GA bulge in helix IV may introduce a structurally conserved kink. We now propose a 'two-domain' structure for the telomerase RNA based on function: one conformationally flexible domain, which includes the template and the region around helix III, involved with enzymatic function, and a second largely helical domain, including helices I and IV and the proposed kink, which may serve as a scaffold for protein binding.

The effects of nucleoside analogs on telomerase and telomeres in Tetrahymena.

The ribonucleoprotein enzyme telomerase is a specialized type of cellular reverse transcriptase which synthesizes one strand of telomeric DNA, using as the template a sequence in the RNA moiety of telomerase. We analyzed the effects of various nucleoside analogs, known to be chain-terminating inhibitors of retroviral reverse transcriptases, on Tetrahymena thermophila telomerase activity in vitro. We also analyzed the effects of such analogs on telomere length and maintenance in vivo, and on vegetative growth and mating of Tetrahymena cells. Arabinofuranyl-guanosine triphosphate (Ara-GTP) and ddGTP both efficiently inhibited telomerase activity in vitro, while azidothymidine triphosphate (AZT-TP), dideoxyinosine triphosphate (ddITP) or ddTTP were less efficient inhibitors. All of these nucleoside triphosphate analogs, however, produced analog-specific alterations of the normal banding patterns seen upon gel electrophoresis of the synthesis products of telomerase, suggesting that their chain terminating and/or competitive actions differ at different positions along the RNA template. The analogs AZT, 3'-deoxy-2',3'-didehydrothymidine (d4T) and Ara-G in nucleoside form caused consistent and rapid telomere shortening in vegetatively growing Tetrahymena. In contrast, ddG or ddI had no effect on telomere length or cell growth rates. AZT caused growth rates and viability to decrease in a fraction of cells, while Ara-G had no such effects even after several weeks in culture. Neither AZT, Ara-G, acycloguanosine (Acyclo-G), ddG nor ddI had any detectable effect on cell mating, as assayed by quantitation of the efficiency of formation of progeny from mated cells. However, AZT decreased the efficiency of programmed de novo telomere addition during macronuclear development in mating cells.