DNA methylation of genes of the main components of the telomerase complex in Danio rerio.

The methylation status of the genes of telomerase reverse transcriptase (tert) and telomerase RNA (terc) was determined in brain tissues of Danio rerio of different age. It is found that, regardless of the age of fish, the regulatory region of the tert gene was completely methylated, whereas the coding region remained unmethylated in all cases. The level of methylation of the region located downstream of the coding region of the terc gene changes with age. This region was analyzed in the samples of other tissues, and its methylation status was also nonuniform. The alteration of the methylation status in the 3'-untranslated region of the terc gene suggests the possibility of transcription of the antisense strand in this region.

Oligonucleotide inhibitors of telomerase: prospects for anticancer therapy and diagnostics.

The activity of telomerase allows eukaryotic cells to have unlimited division potential. On its functioning, telomerase synthesizes short DNA repeats at the 3'-end of DNA within chromosomes that ensures genome stability during cell division. Telomerase is active in the majority of cancer cell types and is virtually absent in somatic cells with rare exceptions. This difference allows us to consider inhibition of telomerase activity as a possible approach to antitumor therapy. Telomerase is a nucleoprotein composed of two main components: the reverse transcriptase (hTERT), which is a catalytic subunit, and telomerase RNA (hTR), which encodes a template for synthesis of repeats. The biogenesis and features of telomerase seem very promising for its inhibition due to complementary interactions. In this review, we analyze putative pathways of oligonucleotide influence on telomerase and consider the known native and modified oligonucleotide inhibitors of telomerase, as well as possible mechanisms of their action. We also discuss the application of telomerase-targeted oligonucleotide conjugates for in vivo imaging of tumor cells.

Structural Features of the Telomerase RNA Gene in the Naked Mole Rat Heterocephalus glaber.

Telomere length, an important feature of life span control, is dependent on the activity of telomerase (a key enzyme of the telomere-length-maintaining system). Telomerase RNA is a component of telomerase and, thus, is crucial for its activity. The structures of telomerase RNA genes and their promoter regions were compared for the long-living naked mole rat and different organisms. Two rare polymorphisms in Heterocephalus glaber telomerase RNA (hgTER) were identified: A→G in the first loop of pseudoknot P2b-p3 (an equivalent of 111nt in hTR) and G→A in the scaRNA domain CR7-p8b (an equivalent of 421nt in hTR). Analysis of TER promoter regions allowed us to identify two new transcription factor binding sites. The first one is the ETS family site, which was found to be a conserved element for all the analyzed TER promoters. The second site is unique for the promoter region of TER of the naked mole rat and is a binding site for the SOX17 transcription factor. The absence of one Sp1 site in the TER promoter region of the naked small rat is an additional specific feature of the promoter area of hgTER. Such variation in the hgTER transcription regulation region and hgTER itself could provide increased telomerase activity in stem cells and an extended lifespan to H. glaber.

Telomerase RNA biosynthesis and processing.

Telomerase synthesizes repetitive G-rich sequences (telomeric repeats) at the ends of eukaryotic chromosomes. This mechanism maintains the integrity of the genome, as telomere shortening leads to degradation and fusion of chromosomes. The core components of telomerase are the telomerase catalytic subunit and telomerase RNA, which possesses a small template region serving for the synthesis of a telomeric repeat. Mutations in the telomerase RNA are associated with some cases of aplastic anemia and also cause dyskeratosis congenita, myelodysplasia, and pulmonary fibrosis. Telomerase is active in 85% of cancers, and telomerase activation is one of the first steps in cell transformation. The study of telomerase and pathways where this enzyme is involved will help to understand the mechanism of the mentioned diseases and to develop new approaches for their treatment. In this review we describe the modern conception of telomerase RNA biosynthesis, processing, and functioning in the three most studied systems - yeast, vertebrates, and ciliates.

Telomere lengthening and other functions of telomerase.

Telomerase is an enzyme that maintains the length of the telomere. The telomere length specifies the number of divisions a cell can undergo before it finally dies (i.e. the proliferative potential of cells). For example, telomerase is activated in embryonic cell lines and the telomere length is maintained at a constant level; therefore, these cells have an unlimited fission potential. Stem cells are characterized by a lower telomerase activity, which enables only partial compensation for the shortening of telomeres. Somatic cells are usually characterized by the absence of telomerase activity. Telomere shortening leads to the attainment of the Hayflick limit, the transition of cells to a state of senescence. The cells subsequently enter a state of crisis, accompanied by massive cell death. The surviving cells become cancer cells, which are capable both of dividing indefinitely and maintaining telomere length (usually with the aid of telomerase). Telomerase is a reverse transcriptase. It consists of two major components: telomerase RNA (TER) and reverse transcriptase (TERT). TER is a non-coding RNA, and it contains the region which serves as a template for telomere synthesis. An increasing number of articles focussing on the alternative functions of telomerase components have recently started appearing. The present review summarizes data on the structure, biogenesis, and functions of telomerase.

Hansenula Polymorpha TERT: A Telomerase Catalytic Subunit Isolated in Recombinant Form with Limited Reverse Transcriptase Activity.

Telomerase is a ribonucleoprotein, the main function of which is to synthesize telomeres, i.e. repetitive sequences which are localized at the ends of eukaryotic chromosomes. Telomerase maintains the stability of the genome in eukaryotic cells by replicating chromosomal ends. The structural and functional investigation of the telomerase complex is significantly restricted due to difficulties connected with the isolation of its main catalytic subunit in recombinant form. Herein, we describe a method developed for the isolation of the recombinant telomerase reverse transcriptase from thermotolerant yeastHansenula polymorpha. A functional test performed for the isolated protein and the RNA/DNA duplex, simulating the interaction of telomerase RNA and telomere, reveals that the isolated catalytic subunit of telomerase possesses limited reverse transcriptase activity.

Telomerase: structure, functions, and activity regulation.

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50-70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.

Telomerase from yeast Saccharomyces cerevisiae is active in vitro as a monomer.

A system for isolation of yeast telomerase via RNA affinity tag in TLC1 RNA was developed. Streptavidin aptamer was inserted at two different positions in TLC1 RNA. Telomerase with TLC1 RNA with one of these inserts is functional in vivo and can be isolated by affinity chromatography in vitro. A telomerase preparation isolated using this technique from a strain producing two distinguishable TLC1 RNA molecules (with and without aptameric insertion) resulted in isolation of active telomerase containing only TLC1 RNA with the aptamer. Our results indicate that yeast telomerase is active in vitro as a monomer.

Telomerase Complex from Yeast Saccharomyces cerevisiae Contains a Biotinylated Component.

Telomerase adds telomeric repeats to single-stranded DNA at the ends of the chromosomes. This enzyme is a ribonucleoprotein complex. Telomerase from yeast Saccharomyces cerevisiae consists of TLC1 RNA, which serves as a template for the synthesis of telomeric repeats, telomerase reverse transcriptase Est2p, and a number of accessory proteins (Est1p, Est3p, Ku70/Ku80, and Sm-complex). We found that the yeast telomerase complex contains a biotinylated component. The telomerase fraction containing biotinylated protein is active in vitro and constitutes a small part of the total amount of active telomerase isolated from cells. We speculate about the nature of the biotinylated component.

Isolation of active yeast telomerase protein Est3p and investigation of its dimerization in vitro.

In this study we proposed a method for isolation of Est3p modified with various affinity tags, which is applicable for structural and functional studies, and investigated homo- and heterodimer formation with various recombinant forms of Est3p.

Saccharomyces cerevisiae telomerase subunit Est3p binds DNA and RNA and stimulates unwinding of RNA/DNA heteroduplexes.

Telomerase is a key participant of telomere length maintenance system in majority of eukaryotes. It synthesizes telomere repeats at 3'-end of telomere DNA according to its own RNA template. In addition to the reverse transcriptase subunit Est2p and telomerase RNA TLC1, yeast telomerase contain Est1p, necessary for telomerase attachment to telomere and telomerase activation, and Est3p, a subunit with unknown function. We have isolated Est3p and examined its biochemical properties. Est3p binds both DNA and RNA oligonucleotides containing telomere repeat sequences and stimulates dissociation of RNA/DNA heteroduplexes. The importance of these properties of Est3p for telomerase function is discussed.

Complex of transfer-messenger RNA and elongation factor Tu. Unexpected modes of interaction.

Transfer-messenger RNA (tmRNA) is a stable RNA in bacteria of 360 +/- 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA. Ribosomes that are stalled either in a coding region of mRNA or at the 3' end of an mRNA fragment lacking a stop codon are rescued by replacing their mRNA for tmRNA. Here we demonstrate that the interaction of tmRNA with the elongation factor Tu shows unexpected features. Deacylated tmRNA can form a complex with either EF-Tu.GDP or EF-Tu.GTP, the association constants are about one order of magnitude smaller than that of an Ala-tRNA.EF-Tu.GTP complex. tmRNA as well as Ala-tmRNA can be efficiently cross-linked with EF-Tu.GDP using a zero-length cross-link. The efficiency of cross-linking in the case of deacylated tmRNA does not depend on an intact CCA-3' end and is about the same, regardless whether protein mixtures such as the post-ribosomal supernatant (S100 enzymes) or purified EF-Tu are present. Two cross-linking sites with EF-Tu.GDP have been identified that are located outside the tRNA part of tmRNA, indicating an unusual interaction of tmRNA with EF-Tu.GDP.

Effect of point mutations at position 89 of the E. coli 5S rRNA on the assembly and activity of the large ribosomal subunit.

Nucleotide residue U89 in the D loop of Escherichia coli 5S rRNA is adjacent to two domains of 23S rRNA in the large ribosomal subunit [Dokudovskaya et al., RNA 2 (1996) 146-152]. 50S ribosomal subunits were reconstituted containing U89(C, G or A) mutants of 5S rRNAs and the activities of the corresponding 70S ribosomes were studied. The U89C mutant behaves similarly to the wild-type 5S rRNA. Replacement of the pyrimidine base at position U89 by more bulky purine bases impairs the incorporation of 5S rRNA into 50S subunits, whereas the particles formed showed full activities in poly(U)-dependent poly(Phe) synthesis in the presence of either U89G or U89A 5S rRNA mutants. The activity of the reconstituted particles depends on the incorporation of 5S rRNA in agreement with early observations.

5S rRNA sugar-phosphate backbone protection in complexes with specific ribosomal proteins.

5S ribosomal RNA forms stable specific complexes with ribosomal proteins L18, L25 and L5. In this work, interaction of phosphate residues of E. coli 5S rRNA within 5S rRNA-protein complexes has been studied. For this purpose 5S rRNA with statistically distributed phosphorothioate residues has been used for complex formation and the accessibility of phosphorothioates to iodine cleavage in the complex and in the free state has been studied. In free 5S rRNA, the phosphate residue at A73 was partially protected, probably due to being involved in the organization of the spatial structure of 5S rRNA. This protection is stronger in the complex with three proteins when the 5S rRNA structure is stabilized. In the 5S rRNA-L18 complex only two phosphate groups, G7 and A34, were protected. L25 in a complex with 5S rRNA protects large numbers of phosphorothioate groups concentrating in two clusters, indicating the possibility of two binding sites for this protein on 5S rRNA. The protection pattern differs from that for individual proteins because of the possible rearrangement of the structure.