Yehuda Tzfati

A Triple Helix within a Pseudoknot Is a Conserved and Essential Element of Telomerase RNA

ABSTRACT Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A·U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C·C+ triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C·U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

Significance Telomeres protect the ends of eukaryotic chromosomes. Telomeres shorten with age and serve as a biological clock that limits cell proliferation. Excessive telomere shortening accelerates aging, but telomere elongation may facilitate cancer. We found inherited mutations in the regulator of telomere elongation helicase 1 (RTEL1), which cause Hoyeraal–Hreidarsson syndrome, a fatal disease characterized by accelerated telomere shortening, immunodeficiency, and developmental defects. Introducing a normal RTEL1 gene into affected cells prevented telomere shortening and extended their lifespan in culture. The telomere defects, genomic instability, and growth arrest observed in RTEL1-deficient cells help in our understanding the central roles of telomeres in aging and cancer. Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

Identification and comparative analysis of telomerase RNAs from Candida species reveal conservation of functional elements

The RNA component of telomerase (telomerase RNA; TER) varies substantially both in sequence composition and size (from approximately 150 nucleotides [nt] to >1500 nt) across species. This dramatic divergence has hampered the identification of TER genes and a large-scale comparative analysis of TER sequences and structures among distantly related species. To identify by phylogenetic analysis conserved sequences and structural features of TER that are of general importance, it is essential to obtain TER sequences from evolutionarily distant groups of species, providing enough conservation within each group and enough variation among the groups. To this end, we identified TER genes in several yeast species with relatively large (>20 base pairs) and nonvariant telomeric repeats, mostly from the genus Candida. Interestingly, several of the TERs reported here are longer than all other yeast TERs known to date. Within these TERs, we predicted a pseudoknot containing U-A.U base triples (conserved in vertebrates, budding yeasts, and ciliates) and a three-way junction element (conserved in vertebrates and budding yeasts). In addition, we identified a novel conserved sequence (CS2a) predicted to reside within an internal-loop structure, in all the budding yeast TERs examined. CS2a is located near the Est1p-binding bulge-stem previously identified in Saccharomyces cerevisiae. Mutational analyses in both budding yeasts S. cerevisiae and Kluyveromyces lactis demonstrate that CS2a is essential for in vivo telomerase function. The comparative and mutational analyses of conserved TER elements reported here provide novel insights into the structure and function of the telomerase ribonucleoprotein complex.

Optical, Electrical and Surface Plasmon Resonance Methods for Detecting Telomerase Activity

Three different sensing platforms for the analysis of telomerase activity in human cells are described. One sensing platform involves the label-free analysis of the telomerase activity by a field-effect-transistor (FET) device. The telomerase-induced extension of a primer associated with the gate of the FET device, in the presence of the nucleotide mixture dNTPs, alters the gate potential, and this allows the detection of telomerase extracted from 65 ± 10 293T (transformed human embryonic kidney) cells/μL. The second sensing platform involves the optical detection of telomerase using CdSe/ZnS quantum dots (QDs). The telomerase-stimulated telomerization of the primer-functionalized QDs in the presence of the nucleotide mixture dNTPs results in the synthesis of the G-rich telomeres. The stacking of hemin on the self-organized G-quadruplexes found on the telomers results in the electron transfer quenching of the QDs, thus providing an optical readout signal. This method enables the detection of telomerase originating from 270 ± 20 293T cells/μL. The third sensing method involves the amplified surface plasmon resonance (SPR) detection of telomerase activity. The telomerization of a primer associated with Au film-coated glass slides, in the presence of telomerase and the nucleotide mixture (dNTPs), results in the formation of telomeres on the surface, and these alter the dielectric properties of the surface resulting in a shift in the SPR spectrum. The hybridization of Au NPs functionalized with nucleic acids complementary to the telomere repeat units with the telomeres amplifies the SPR shifts due to the coupling between the local plasmon of the NPs and the surface plasmon wave. This method enables the detection of telomerase extracted from 18 ± 3 293T cells/μL.

DNAzyme-like activity of hemin-telomeric G-quadruplexes for the optical analysis of telomerase and its inhibitors.

The telomeres cap and protect the eukaryotic chromosome ends from undesired degradation and end-to-end fusion. They consist of short tandem DNA repeats (TTAGGG in vertebrates). In human somatic cells, the telomeres undergo progressive shortening during cell proliferation, and at a certain length they signal the cell to terminate its life cycle and undergo apoptosis. Telomerase is a ribonucleoprotein reverse transcriptase. It binds to the telomere ends and elongates them by copying telomeric repeats from its endogenous RNA template. This telomerase-mediated elongation of the telomeres compensates for the natural shortening of the telomeres, thus prolonging cellular lifespan and potentially transforming the cell into a malignant form. Indeed, over 85 % of all cancer cells exhibit elevated amounts of telomerase. Accordingly, the rapid, cost-effective, easy, and sensitive analysis of telomerase activity is important for diagnosis and for the development of anticancer drugs (telomerase inhibitors). Various analytical methods have been developed to analyze the activity of telomerase. The most frequently used method is the telomeric repeat amplification protocol (TRAP), which is PCR-based. However, this method is susceptible to inhibition of the PCR by the cell extract, requires costly instrumentation and reagents, and is time consuming. Moreover, the exponential amplification of the telomerase products increases the risk of false-positive results, and makes the quantitative analysis of telomerase products difficult. Various electrochemical and optical methods to monitor telomerase activity have been reported. Electrochemical methods include the electrochemical detection of ferrocenyl naphthalene diimide, which binds to the telomeric tetraplex structure, and the coulometric analysis of the Ru(NH3)6 3+ label, which binds to duplex DNA formed between the telomeric repeats and a nucleic acid complementary to the telomere repeat units. Optical methods for detection of telomerase activity include the fluorescence resonance energy transfer (FRET) process between quantum dots and an acceptor dye incorporated into telomeres synthesized by telomerase on the quantum dots. The detection of telomerase activity has also been carried out by designing DNA hairpin structures that include a protected horseradish peroxidase mimicking DNAzyme structure in the stem region. Activation of the DNAzyme stimulates a colored reaction upon opening of the hairpins by the telomeres. Nanotechnology-based detection of telomeres by magnetomechanical deflection of cantilevers has been reported. Activation of telomerase activity on the cantilevers in combination with specific hybridization of magnetic particles functionalized with nucleic acids complementary to the telomere repeat units enables forced deflection of the cantilever under an applied magnetic field. Although substantial progress has been achieved in the analysis of telomerase activity, the sensitivity of the various platforms is usually insufficient and requires a preceding PCR amplification step, which is accompanied by the aforementioned drawbacks. In the presence of hemin (1), G-rich DNA sequences are known to form catalytic hemin–G-quadruplex nanostructures. The best known system is the horseradish peroxidase mimicking hemin–G-quadruplex that was found to catalyze the H2O2-mediated oxidation of 2,2’-azinobis(3-ethylbenzthiazoline6-sulfonic acid), ABTS , to the colored product, ABTSC , or to catalyze the generation of chemiluminescence in the presence of luminol/H2O2. In fact, many sensing platforms have used the hemin–G-quadruplex DNAzyme as a catalytic label. Further studies have demonstrated that various G-rich DNA sequences which self-assemble into parallel or antiparallel G-quadruplexes reveal horseradish-peroxidase-like catalytic activities in the presence of hemin. The relative activities of the hemin–Gquadruplexes are dominated by the specific configuration of the nanostructures. The G-rich telomeric repeat units are known to adopt a G-quadruplex conformation, and are therefore anticipated to incorporate hemin in catalytically active structures. These could then serve as biocatalytic amplifying labels that follow telomerase activity. In fact, several studies have reported the catalytic activities of the complex generated between hemin and a synthetic DNA strand that includes the telomeric repeat sequence, but this phenomenon was never implemented to detect telomerase activities in biological samples. Herein we report the development of a colorimetric assay that allows quantitative monitoring of telomerase activity originating from transformed human embryonic kidney (293T) cells, and to probe inhibitors of telomerase. Hemin inhibits the activity of telomerase through stabilization of the G-quadruplex structure of the telomere ends, and we found that the cell extract perturbs the hemin–G-quadruplex DNAzyme functions. Nonetheless, these difficulties were resolved by the appropriate design of the analytical steps, and a colorimetric [a] R. Freeman, E. Sharon, Dr. C. Teller, A. Henning , Prof. I. Willner Institute of Chemistry, The Center for Nanoscience and Nanotechnology The Hebrew University of Jerusalem, Jerusalem 91904 (Israel) Fax: (+ 972) 2-6527715 E-mail : willnea@vms.huji.ac.il [b] Dr. C. Teller Fraunhofer Institute for Biomedical Engineering IBMT Am M hlenberg 13, 14476 Potsdam-Golm (Germany) [c] A. Henning Physikalische Chemie, Messund Sensortechnik Technische Universit t Dresden, 01062 Dresden (Germany) [d] Dr. Y. Tzfati Department of Genetics, The Silberman Institute of Life Sciences The Hebrew University of Jerusalem, Jerusalem 91904 (Israel) Fax: (+ 972) 2-6584902 Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000512.

A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs

The telomerase ribonucleoprotein copies a short template within its integral RNA moiety onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Non-template regions of telomerase RNA (TER) are also crucial for telomerase function, yet they are highly divergent in sequence among species and their roles are largely unclear. Using both phylogenetic and mutational analyses, we predicted secondary structures for TERs from Kluyveromyces budding yeast species. A comparison of these secondary structure models with the published model for the Saccharomyces cerevisiae TER reveals a common arrangement into three long arms, a templating domain in the center and several conserved elements in the same positions within the structure. One of them, a three-way junction element, is highly conserved in budding yeast TERs. This element also shows sequence and structure similarity to the critical CR4-CR5 activating domain of vertebrate TERs. Mutational analysis in Kluyveromyces lactis confirmed that this element, and in particular the residues conserved across yeast and vertebrates, is critical for telomerase action both in vivo and in vitro. These findings demonstrate that despite the extreme divergence of TER sequences from different organisms, they do share conserved elements, which presumably carry out common roles in telomerase function.

Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids PURIFICATION AND CHARACTERIZATION

Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5′-GGGGTTGGTGTA-3′). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not double-stranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.

Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo

Telomerase is a ribonucleoprotein complex that extends the 3′ ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C+•G-C triple adjacent to a stem 2–loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.

Unraveling the pathogenesis of Hoyeraal–Hreidarsson syndrome, a complex telomere biology disorder

Hoyeraal–Hreidarsson (HH) syndrome is a multisystem genetic disorder characterized by very short telomeres and considered a clinically severe variant of dyskeratosis congenita. The main cause of mortality, usually in early childhood, is bone marrow failure. Mutations in several telomere biology genes have been reported to cause HH in about 60% of the HH patients, but the genetic defects in the rest of the patients are still unknown. Understanding the aetiology of HH and its diverse manifestations is challenging because of the complexity of telomere biology and the multiple telomeric and non‐telomeric functions played by telomere‐associated proteins in processes such as telomere replication, telomere protection, DNA damage response and ribosome and spliceosome assembly. Here we review the known clinical complications, molecular defects and germline mutations associated with HH, and elucidate possible mechanistic explanations and remaining questions in our understanding of the disease.

Electron-transfer quenching of nucleic acid-functionalized CdSe/ZnS quantum dots by doxorubicin: A versatile system for the optical detection of DNA, aptamer–substrate complexes and telomerase activity

The optical detection of DNA or the sensing of low-molecular-weight substrates or proteins by aptamer nucleic acids is a long term challenge in the design of biosensors. Similarly, the detection of the telomerase activity, a versatile biomarker of cancer cells, is important for rapid cancer diagnostics. We implement the luminescence quenching of the CdSe/ZnS quantum dots (QDs) as a versatile process to develop DNA sensors and aptasensors, and to design an analytical platform for the detection of telomerase activity. The formation of nucleic acid duplexes on QDs, or the assembly of aptamer-substrate complexes on the QDs (substrate=cocaine or thrombin) is accompanied by the intercalation of doxorubicin (DB) into the duplex domains of the resulting recognition complexes. The intercalated DB quenches the luminescence of the QDs, thus leading to the detection readout signal. Similarly, the telomerase-induced formation of the telomere chains on the QDs is followed by the hybridization of nucleic-acid units complementary to the telomere repeat units, and the intercalation of DB into the resulting duplex structure. The resulting luminescence quenching of the QDs provides an indicating signal for the activity of telomerase.