RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

Telomerase synthesizes telomeric DNA repeats onto chromosome termini from an intrinsic RNA template. The processive synthesis of DNA repeats relies on a unique, yet poorly understood, mechanism whereby the telomerase RNA template translocates and realigns with the DNA primer after synthesizing each repeat. Here, we provide evidence that binding of the realigned RNA/DNA hybrid by the active site is an essential step for template translocation. Employing a template-free human telomerase system, we demonstrate that the telomerase active site directly binds to RNA/DNA hybrid substrates for DNA polymerization. In telomerase processivity mutants, the template-translocation efficiency correlates with the affinity for the RNA/DNA hybrid substrate. Furthermore, the active site is unoccupied during template translocation as a 5 bp extrinsic RNA/DNA hybrid effectively reduces the processivity of the template-containing telomerase. This suggests that strand separation and template realignment occur outside the active site, preceding the binding of realigned hybrid to the active site. Our results provide new insights into the ancient RNA/DNA hybrid binding ability of telomerase and its role in template translocation.     

Elucidation of the mode of interaction in the UP1-telomerase RNA-telomeric DNA ternary complex which serves to recruit telomerase to telomeric DNA and to enhance the telomerase activity

We found that UP1, a proteolytic product of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), both enhances and represses the telomerase activity. The formation of the UP1–telomerase RNA–telomeric DNA ternary complex was revealed by a gel retardation experiment. The interactions in the ternary and binary complexes were elucidated by NMR. UP1 has two nucleic acid-binding domains, BD1 and BD2. In the UP1–telomerase RNA binary complex, both BD1 and BD2 interact with telomerase RNA. Interestingly, when telomeric DNA was added to the binary complex, telomeric DNA bound to BD1 in place of telomerase RNA. Thus, BD1 basically binds to telomeric DNA, while BD2 mainly binds to telomerase RNA, which resulted in the formation of the ternary complex. Here, UP1 bridges telomerase and telomeric DNA. It is supposed that UP1/hnRNP A1 serves to recruit telomerase to telomeric DNA through the formation of the ternary complex. A model has been proposed for how hnRNP A1/UP1 contributes to enhancement of the telomerase activity through recruitment and unfolding of the quadruplex of telomeric DNA.     

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G‐rich sequence, and with noncoding RNA, Telomeric repeat‐containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2′‐OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G‐quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate – the 3′‐end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3′‐end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Telomerase RNA localized in the replication band and spherical subnuclear organelles in hypotrichous ciliates

The intranuclear distribution of telomere DNA-binding protein and telomerase RNA in hypotrichous ciliates was revealed by indirect fluorescent antibody staining and in situ hybridization. The Oxytricha telomere protein colocalized with DNA, both being dispersed throughout the macronucleus except for numerous spherical foci that contained neither DNA nor the protein. Surprisingly, the telomerase RNA was concentrated in these foci; therefore, much of telomerase does not colocalize with telomeres. These foci persist through the cell cycle. They may represent sites of assembly, transport or stockpiling of telomerase and other ribonucleoproteins. During S phase, the macronuclear DNA replication machinery is organized into a disc-shaped structure called the replication band. Telomerase RNA is enriched in the replication band as judged by fluorescence intensity. We conclude that the localization of a subfraction of telomerase is coordinated with semiconservative DNA replication.     

Non-telomeric activities of telomerase

Recent results suggest that telomerase is involved in many more cellular processes than merely telomere elongation. These include telomere-independent anti-apoptotic, cytoprotective and pro-proliferative effects of telomerase or protection of mitochondrial DNA against oxidative stress. Telomerase also participates in DNA repair and its essential subunits, hTR and hTERT, are able to modulate independently the cell's response to DNA damage. Recent high throughput analyses of gene expression showed that hTERT expression modulates expression of about 300 genes, including genes involved in the regulation of cell cycle progression, proliferation and differentiation. Besides the well-known telomerase catalytic activity of RNA-dependent DNA polymerase, its RNA-dependent RNA polymerase activity was recently described in association with the RNA subunit of mitochondrial RNA processing endoribonuclease, thus suggesting involvement of telomerase in RNA interference processes. These recent discoveries open novel possibilities and entirely unexpected research perspectives, branching off from the mainstream telomere and telomerase research.     

Tetrahymena telomerase RNA levels increase during macronuclear development.

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000-40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2- to 5-fold at 9-15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells.     

The Est1 subunit of yeast telomerase binds the Tlc1 telomerase RNA

Est1 is a component of yeast telomerase, and est1 mutants have senescence and telomere loss phenotypes. The exact function of Est1 is not known, and it is not homologous to components of other telomerases. We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity. Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1. Deletion of EBS1 results in short telomeres. We created point mutations in a putative RRM of Est1. One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants. Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA. Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA. Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM. Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.     

Developmentally programmed healing of chromosomes by telomerase in Tetrahymena

Healing of a broken chromosome and in eukaryotes involves acquisition of a telomere. During macronuclear development in ciliated protozoans, germline chromosomes are fragmented into linear subchromosomes, whose ends are healed by de novo addition of telomeres. We showed previously that the ribonucleoprotein enzyme telomerase elongates preexisting telomeres by synthesizing one telomeric DNA strand, using a template sequence in the RNA moiety of the enzyme. By marking telomerase with a mutation in the telomerase RNA template, which causes synthesis of novel telomeric sequences, we now show that in the ciliate Tetrahymena, telomerase directly adds telomeric DNA onto nontelomeric sequences during developmentally controlled chromosome healing. Unexpectedly, one telomerase RNA template mutation converted telomerase from an enzyme that normally synthesizes precisely templated sequences to a less precise polymerase that sometimes synthesizes irregular telomeric repeats in vivo.     

Circular rDNA Replicons Persist in Tetrahymena thermophila Transformants Synthesizing GGGGTC Telomeric Repeats

Site-directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo. The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase RNA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection. Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation.     

鸡端粒酶RNA基因的克隆

本研究采用扩增条件优化的PCR扩增技术,以MDCC-MSBl细胞基因组DNA为模板扩增出鸡端粒酶RNA(chicken telomerase RNA,chTR)全长基因,克隆到pMD18-T载体中,经酶切鉴定和PCR鉴定后测定序列.序列分析表明所克隆的鸡端粒酶RNA基因全长465 bp,其中模板区的11个核苷(5'-CUAACCCUAAU-3')合成端粒亚单位(TTAGGG)n.chTR基因的克隆为进一步研究chTR在马立克氏病发病过程中的作用以及马立克氏病的发病机制提供可能的序列基础.     

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.     

Sequence-specific DNA primer effects on telomerase polymerization activity.

The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo.