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The germline-soma divide is a fundamental distinction in developmental biology, and different genes are expressed in germline and somatic cells throughout metazoan life cycles. Ciliates, a group of microbial eukaryotes, exhibit germline-somatic nuclear dimorphism within a single cell with two different genomes. The ciliate Oxytricha trifallax undergoes massive RNA-guided DNA elimination and genome rearrangement to produce a new somatic macronucleus (MAC) from a copy of the germline micronucleus (MIC). This process eliminates noncoding DNA sequences that interrupt genes and also deletes hundreds of germline-limited open reading frames (ORFs) that are transcribed during genome rearrangement. Here, we update the set of transcribed germline-limited ORFs (TGLOs) in O. trifallax. We show that TGLOs tend to be expressed during nuclear development and then are absent from the somatic MAC. We also demonstrate that exposure to synthetic RNA can reprogram TGLO retention in the somatic MAC and that TGLO retention leads to transcription outside the normal developmental program. These data suggest that TGLOs represent a group of developmentally regulated protein-coding sequences whose gene expression is terminated by DNA elimination., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

G-quadruplexes (GQs) are highly stable noncanonical forms of nucleic acids that are present in important genomic regions. The central core of the GQ is lined up by four closely spaced carbonyl groups from the G-quartets, and the resulting electrostatic repulsion is neutralized by the coordinating cations. In spite of several reports on GQ structure and cation-GQ interactions, the atomic- to molecular-level understanding of the ion dynamics and ion exchange in the GQ core is quite poor. Here, we attempt to elucidate the mechanism of Na + and K + binding to the GQ core and trace the exchange of these ions with the ions in bulk by means of all-atomic molecular dynamics (MD) simulations. One of the most studied GQs, Oxytricha nova telomeric G-quadruplex (OxyGQ), is taken as the representative GQ. Subsequently, umbrella sampling MD simulations were performed to elucidate the energetics of ion translocation from one end to the other end of the GQ central core. Our study highlights the importance of ion hydration for the uptake and correct positioning of the cations in the core. The free-energy landscape of ion transport has shown favorable in-plane binding of Na + ions with GQ quartets, which matches very well with the crystal structure. The binding of K + ions, on the other hand, was out-of-plane and its translocation required a larger barrier to cross.

DNA recombinant processes can involve gene segments that overlap or interleave with gene segments of another gene. Such gene segment appearances relative to each other are called here gene segment organization. We use graphs to represent the gene segment organization in a chromosome locus. Vertices of the graph represent contigs resulting after the recombination and the edges represent the gene segment organization prior to rearrangement. To each graph we associate a vector whose entries correspond to graph properties, and consider this vector as a point in a higher dimensional Euclidean space such that cluster formations and analysis can be performed with a hierarchical clustering method. The analysis is applied to a recently sequenced model organism Oxytricha trifallax, a species of ciliate with highly scrambled genome that undergoes massive rearrangement process after conjugation. The analysis shows some emerging star-like graph structures indicating that segments of a single gene can interleave, or even contain all of the segments from fifteen or more other genes in between its segments. We also observe that as many as six genes can have their segments mutually interleaving or overlapping., (Copyright © 2020. Published by Elsevier Ltd.)

Extrachromosomal circular DNA (eccDNA) is both a driver of eukaryotic genome instability and a product of programmed genome rearrangements, but its extent had not been surveyed in Oxytricha, a ciliate with elaborate DNA elimination and translocation during development. Here, we captured rearrangement-specific circular DNA molecules across the genome to gain insight into its processes of programmed genome rearrangement. We recovered thousands of circularly excised Tc1/mariner-type transposable elements and high confidence non-repetitive germline-limited loci. We verified their bona fide circular topology using circular DNA deep-sequencing, 2D gel electrophoresis and inverse polymerase chain reaction. In contrast to the precise circular excision of transposable elements, we report widespread heterogeneity in the circular excision of non-repetitive germline-limited loci. We also demonstrate that circular DNAs are transcribed in Oxytricha, producing rearrangement-specific long non-coding RNAs. The programmed formation of thousands of eccDNA molecules makes Oxytricha a model system for studying nucleic acid topology. It also suggests involvement of eccDNA in programmed genome rearrangement., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ciliate Oxytricha trifallax contains two nuclei: a germline micronucleus and a somatic macronucleus. These two nuclei diverge significantly in genomic structure. The micronucleus contains approximately 100 chromosomes of megabase scale, while the macronucleus contains 16,000 gene-sized, high ploidy "nanochromosomes." During its sexual cycle, a copy of the zygotic germline micronucleus develops into a somatic macronucleus via DNA excision and rearrangement. The rearrangement process is guided by multiple RNA-based pathways that program the epigenetic inheritance of sequences in the parental macronucleus of the subsequent generation. Here, we show that the introduction of synthetic DNA molecules homologous to a complete native nanochromosome during the rearrangement process results in either loss or heavy copy number reduction of the targeted nanochromosome in the macronucleus of the subsequent generation. This phenomenon was tested on a variety of nanochromosomes with different micronuclear structures, with deletions resulting in all cases. Deletion of the targeted nanochromosome results in the loss of expression of the targeted genes, including gene knockout phenotypes that were phenocopied using alternative knockdown approaches. Further investigation of the chromosome deletion showed that, although the full length nanochromosome was lost, remnants of the targeted chromosome remain. We were also able to detect the presence of telomeres on these remnants. The chromosome deletions and remnants are epigenetically inherited when backcrossed to wild type strains, suggesting that an undiscovered mechanism programs DNA elimination and cytoplasmically transfers to both daughter cells during conjugation. Programmed deletion of targeted chromosomes provides a novel approach to investigate genome rearrangement and expands the available strategies for gene knockout in Oxytricha trifallax ., (Copyright © 2019 Clay et al.)

Oxytricha trifallax , like other ciliates, has separate germline and somatic nuclei. The diploid germline genome in the micronucleus is composed of long conventional chromosomes. The macronucleus contains a somatic genome which is naturally fragmented into thousands of kilobase-sized chromosomes. Here, we develop a method to stably incorporate artificial chromosomes into the macronucleus. We report two cases of successful transformation and demonstrate the use of somatic transformation to investigate gene regulation and gene function in Oxytricha We show that the transformed artificial chromosomes are maintained through multiple asexual divisions. Furthermore, they support the transcriptional regulation of the native chromosome from which they were derived and are translated to produce functional proteins. To test if transformed chromosomes are amenable to practical applications, we generated a tagged version of a representative gene (AL1) and used it to co-precipitate associated proteins. This revealed an association with nucleic acid binding proteins, specifically RNA-binding proteins, and RNA immunoprecipitation of AL1 revealed its association with multiple RNAs. The use of artificial chromosomes in Oxytricha enables an array of genetic and molecular biological assays, as well as new avenues of inquiry into the epigenetic programming of macronuclear development and genome rearrangement., (Copyright © 2019 Clay et al.)

A new hypotrichous ciliate, Oxytricha seokmoensis sp. nov., was discovered in a soil from a forest in South Korea and described based on the observations of living and stained specimens. In addition, phylogenetic analyses were performed using the small subunit ribosomal RNA (18S rRNA) gene sequence. Morphologically, the new species is similar to the O. granulifera-complex in terms of ciliary structure and arrangement of cortical granules, but dorsal kineties 3 and 4 (not completely separated vs. separated) and macronuclear nodules in the cyst (separated vs. fused) differ. Oxytricha seokmoensis is most similar to O. pulvillus, but can be distinguished by the number of adoral membranelles (30-40 vs. 23-27), contractile vacuole (present vs. absent), number of left (27-37 vs. 17-25) and right (27-35 vs. 18-23) marginal cirri, and lepidosomes on the cyst surface (present vs. absent). In a phylogenetic tree, O. seokmoensis is distinctly separated from the O. granulifera clade, but is sister to the Paroxytricha clade. In addition, O. seokmoensis and P. longigranulosa have the smallest genetic difference (d = 0.015, 23 of 1579 nt difference). This close relationship is supported by incomplete dorsal kinety 3 fragmentation and separated macronuclear nodules in resting cysts., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Conventional assessment and evaluation of sediment quality are based on laboratory-based ecotoxicological and chemical measurements with lack of concern for ecological relevance. Microbiotas in sediment are responsive to pollutants and can be used as alternative ecological indicators of sediment pollutants; however, the linkage between the microbial ecology and ecotoxicological endpoints in response to sediment contamination has been poorly evaluated. Here, in situ microbiotas from the Three Gorges Reservoir (TGR) area of the Yangtze River were characterized by DNA metabarcoding approaches, and then, changes of in situ microbiotas were compared with the ecotoxicological endpoint, aryl hydrocarbon receptor (AhR) mediated activity, and level of polycyclic aromatic hydrocarbons (PAHs) in sediments. PAHs and organic pollutant mixtures mediating AhR activity had different effects on the structures of microbiotas. Specifically, Shannon indices of protistan communities were negatively correlated with the levels of AhR mediated activity and PAHs. The sediment AhR activity was positively correlated with the relative abundance of prokaryotic Acetobacteraceae, but had a negative correlation with protistan Oxytrichidae. Furthermore, a quantitative classification model was built to predict the level of AhR activity based on the relative abundances of Acetobacteraceae and Oxytrichidae. These results suggested that in situ Protista communities could provide a useful tool for monitoring and assessing ecological stressors. The observed responses of microbial community provided supplementary evidence to support that the AhR-active pollutants, such as PAHs, were the primary stressors of the aquatic community in TGR area., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a germline nucleus (micronucleus; abbr. MIC). Furthermore, Oxytricha trifallax undergoes extensive genome rearrangements during sexual conjugation and post-zygotic development of daughter cells. These rearrangements are necessary because the precursor MIC loci are often both fragmented and scrambled, with respect to the corresponding MAC loci. Such genome architectures are remarkably tolerant of encrypted MIC loci, because RNA-guided processes during MAC development reorganize the gene fragments in the correct order to resemble the parental MAC sequence. Here, we describe the germline organization of several nested and highly scrambled genes in Oxytricha trifallax These include cases with multiple layers of nesting, plus highly interleaved or tangled precursor loci that appear to deviate from previously described patterns. We present mathematical methods to measure the degree of nesting between precursor MIC loci, and revisit a method for a mathematical description of scrambling. After applying these methods to the chromosome rearrangement maps of O. trifallax we describe cases of nested arrangements with up to five layers of embedded genes, as well as the most scrambled loci in O. trifallax ., (Copyright © 2018 Braun et al.)

The genus Oxytricha Bory de Saint-Vincent in Lamouroux, Bory de Saint-Vincent and Deslongchamps, 1824 comprises about 38 species distributed worldwide and has been considered to be a nonmonophyletic group. Based on living observations, protargol preparations, and a small subunit ribosomal RNA (SSU rRNA) gene sequence, we describe a new subspecies Oxytricha granulifera chiapasensis n. subsp. This new taxon is morphologically characterized by undulating membranes basically in a Stylonychia-pattern, six dorsal kineties, size in vivo ca. 60-120 × 20-40 μm, 21-30 right and 21-31 left marginal cirri, 22-29 adoral membranelles, and spherical cortical granules arranged in longitudinal rows on the dorsal side. In terms of the SSU rRNA gene sequence, the new subspecies differs from populations of O. granulifera from GENBANK by 7-35 nucleotides. Phylogenetic analyses showed that Oxytricha granulifera gene sequences were nested into three groups, with the new subspecies included in one of them. Oxytricha granulifera chiapasensis n. subsp. is different from Oxytricha granulifera granulifera Foissner and Adam, 1983 and Oxytricha granulifera quadricirrata Blatterer and Foissner, 1988 based on: (i) undulating membranes in Stylonychia-pattern, (ii) formation of a sixth dorsal kinety during morphogenesis, (iii) the adoral membranelles number, and (iv) inhabiting freshwater habitats., (© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.)

Dicer-dependent small noncoding RNAs play important roles in gene regulation in a wide variety of organisms. Endogenous small interfering RNAs (siRNAs) are part of an ancient pathway of transposon control in plants and animals. The ciliate, Oxytricha trifallax, has approximately 16,000 gene-sized chromosomes in its somatic nucleus. Long noncoding RNAs establish high ploidy levels at the onset of sexual development, but the factors that regulate chromosome copy numbers during cell division and growth have been a mystery. We report a novel function of a class of Dicer (Dcl-1)- and RNA-dependent RNA polymerase (RdRP)-dependent endogenous small RNAs in regulating chromosome copy number and gene dosage in O. trifallax Asexually growing populations express an abundant class of 21-nt sRNAs that map to both coding and noncoding regions of most chromosomes. These sRNAs are bound to chromatin and their levels surprisingly do not correlate with mRNA levels. Instead, the levels of these small RNAs correlate with genomic DNA copy number. Reduced sRNA levels in dcl-1 or rdrp mutants lead to concomitant reduction in chromosome copy number. Furthermore, these cells show no signs of transposon activation, but instead display irregular nuclear architecture and signs of replication stress. In conclusion, Oxytricha Dcl-1 and RdRP-dependent small RNAs that derive from the somatic nucleus contribute to the maintenance of gene dosage, possibly via a role in DNA replication, offering a novel role for these small RNAs in eukaryotes., (© 2018 Khurana et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The ciliate Oxytricha trifallax maintains two genomes: a germline genome that is active only during sexual conjugation and a transcriptionally active, somatic genome that derives from the germline via extensive sequence reduction and rearrangement. Previously, we found that long noncoding (lnc) RNA "templates"-telomere-containing, RNA-cached copies of mature chromosomes-provide the information to program the rearrangement process. Here we used a modified RNA-seq approach to conduct the first genome-wide search for endogenous, telomere-to-telomere RNA transcripts. We find that during development, Oxytricha produces long noncoding RNA copies for over 10,000 of its 16,000 somatic chromosomes, consistent with a model in which Oxytricha transmits an RNA-cached copy of its somatic genome to the sexual progeny. Both the primary sequence and expression profile of a somatic chromosome influence the temporal distribution and abundance of individual template RNAs. This suggests that Oxytricha may undergo multiple rounds of DNA rearrangement during development. These observations implicate a complex set of thousands of long RNA molecules in the wiring and maintenance of a highly elaborate somatic genome architecture., (© 2017 Lindblad et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines.

Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

During its sexual reproduction, the stichotrichous ciliate Oxytricha trifallax orchestrates a remarkable transformation of one of the newly formed germline micronuclear genomes. Hundreds of thousands of gene pieces are stitched together, excised from chromosomes, and replicated dozens of times to yield a functional somatic macronuclear genome composed of ~16,000 distinct DNA molecules that typically encode a single gene. Little is known about the proteins that carry out this process. We profiled mRNA expression as a function of macronuclear development and identified hundreds of mRNAs preferentially expressed at specific times during the program. We find that a disproportionate number of these mRNAs encode proteins that are involved in DNA and RNA functions. Many mRNAs preferentially expressed during macronuclear development have paralogs that are either expressed constitutively or are expressed at different times during macronuclear development, including many components of the RNA polymerase II machinery and homologous recombination complexes. Hundreds of macronuclear development-specific genes encode proteins that are well-conserved among multicellular eukaryotes, including many with links to germline functions or development. Our work implicates dozens of DNA and RNA-binding proteins with diverse evolutionary trajectories in macronuclear development in O. trifallax. It suggests functional connections between the process of macronuclear development in unicellular ciliates and germline specialization and differentiation in multicellular organisms, and argues that gene duplication is a key source of evolutionary innovation in this process.

Some genera of ciliates, such as Oxytricha and Stylonychia, undergo massive genome reorganization during development and provide model organisms to study DNA rearrangement. A common feature of these ciliates is the presence of two types of nuclei: a germline micronucleus and a transcriptionally-active somatic macronucleus containing over 16,000 gene sized "nano-chromosomes". During conjugation the old parental macronucleus disintegrates and a new macronucleus forms from a copy of the zygotic micronucleus. During this process, macronuclear chromosomes assemble through DNA processing events that delete 90-98% of the DNA content of the micronucleus. This includes the deletion of noncoding DNA segments that interrupt precursor DNA regions in the micronucleus, as well as transposons and other germline-limited DNA. Each macronuclear locus may be present in the micronucleus as several nonconsecutive, permuted, and/or inverted DNA segments. Here we investigate the genome-wide range of scrambled gene architectures that describe all precursor-product relationships in Oxytricha trifallax, the first completely sequenced scrambled genome. We find that five general, recurrent patterns in the sets of scrambled micronuclear precursor pieces can describe over 80% of Oxytricha's scrambled genes. These include instances of translocations and inversions, and other specific patterns characterized by alternating stretches of consecutive odd and even DNA segments. Moreover, we find that iterating patterns of alternating odd-even segments up to four times can describe over 96% of the scrambled precursor loci. Recurrence of these highly structured genetic architectures within scrambled genes presumably reflects recurrent evolutionary events that gave rise to over 3000 of scrambled loci in the germline genome., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The genome is often described as the information repository of an organism. Whether millions or billions of letters of DNA, its transmission across generations confers the principal medium for inheritance of organismal traits. Several emerging areas of research demonstrate that this definition is an oversimplification. Here, we explore ways in which a deeper understanding of genomic diversity and cell physiology is challenging the concepts of physical permanence attached to the genome as well as its role as the sole information source for an organism.

Ciliated protists exhibit nuclear dimorphism through the presence of somatic macronuclei (MAC) and germline micronuclei (MIC). In some ciliates, DNA from precursor segments in the MIC genome rearranges to form transcriptionally active genes in the mature MAC genome, making these ciliates model organisms to study the process of somatic genome rearrangement. Similar broad scale, somatic rearrangement events occur in many eukaryotic cells and tumors. The (http://oxytricha.princeton.edu/mds_ies_db) is a database of genome recombination and rearrangement annotations, and it provides tools for visualization and comparative analysis of precursor and product genomes. The database currently contains annotations for two completely sequenced ciliate genomes: Oxytricha trifallax and Tetrahymena thermophila., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dynein heavy chains are motor proteins that comprise a large gene family found across eukaryotes. We have investigated this gene family in four ciliate species: Ichthyophthirius, Oxytricha, Paramecium, and Tetrahymena. Ciliates appear to encode more dynein heavy chain genes than most eukaryotes. Phylogenetic comparisons demonstrated that the last common ancestor of the ciliates that were examined expressed at least 14 types of dynein heavy chains with most of the expansion coming from the single-headed inner arm dyneins. Each of the dyneins most likely performed different functions within the cell., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ∼130 μs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-μs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include non-specific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g. structure the propeller loops., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Programmed genome rearrangements in the unicellular eukaryote Oxytricha trifallax produce a transcriptionally active somatic nucleus from a copy of its germline nucleus during development. This process eliminates noncoding sequences that interrupt coding regions in the germline genome, and joins over 225,000 remaining DNA segments, some of which require inversion or complex permutation to build functional genes. This dynamic genomic organization permits some single DNA segments in the germline to contribute to multiple, distinct somatic genes via alternative processing. Like alternative mRNA splicing, the combinatorial assembly of DNA segments contributes to genetic variation and facilitates the evolution of new genes. In this study, we use comparative genomic analysis to demonstrate that the emergence of alternative DNA splicing is associated with the origin of new genes. Short duplications give rise to alternative gene segments that are spliced to the shared gene segments. Alternative gene segments evolve faster than shared, constitutive segments. Genes with shared segments frequently have different expression profiles, permitting functional divergence. This study reports alternative DNA splicing as a mechanism of new gene origination, illustrating how the process of programmed genome rearrangement gives rise to evolutionary innovation., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Several guanine-rich sequences exist in many important regions, such as telomeres, and these sequences can form quadruplex DNA structures. It was previously reported that 3'-guanines are mainly oxidized in the Tetrahymena and Oxytricha telomeric quadruplex DNA, d(TGGGGT)4, and 5'-guanines are mainly oxidized in the human telomeric quadruplex DNA, d(TAGGGT)4T. We speculated that the differences in site reactivity between d(TGGGGT)4 and d(TAGGGT)4T are induced by the localization of the HOMO. The HOMOs of the possible quadruplex structures were thus determined and the results showed that the HOMOs of d(TGGGGT)4 +3K(+) and d(TAGGGT)4T +2K(+) localized at the 5'-guanine, and that the HOMO shifted from the 5'-guanine to the 3'-guanine by the addition of a 5'-capping cation. Furthermore, we determined the influence of the cation and demonstrated that localization of the HOMO at the G-quartet plane located immediately adjacent to the cation is disfavored. The calculated HOMO localization of d(TGGGGT)4 +4K(+) and d(TAGGGT)4T +2K(+) matched the experimental results and suggest that d(TGGGGT)4 contains a 5'-capping cation in solution., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Guanine (G)-rich DNA sequences can adopt four-stranded quadruplex conformations that may play a role in the regulation of genetic processes. To explore the possibility of targeted molecular recognition of DNA sequences with short G-rich peptide nucleic acids (PNA) and to assess the strand arrangement in such complexes, we used PNA and DNA with the Oxytricha nova telomeric sequence d(G4T4G4) as a model. PNA probes were complexed with DNA targets in the following forms: single-stranded oligonucleotides, a loop of DNA in a hairpin conformation, and as supercoiled plasmid with the (G4T4G4)/(C4A4C4) insert. Gel-shift mobility assays demonstrated formation of stable hybrid complexes between the homologous G4T4G4 PNA and DNA with multiple modes of binding. Chemical and enzymatic probing revealed sequence-specific and G-quadruplex dependent binding of G4T4G4 PNA to dsDNA. Spectroscopic and electrophoretic analysis of the complex formed between PNA and the synthetic DNA hairpin containing the G4T4G4 loop showed that the stoichiometry of a prevailing complex is three PNA strands per one DNA strand. We speculate how this new PNA-DNA complex architecture can help to design more selective, quadruplex-specific PNA probes.

The ciliate Oxytricha is a microbial eukaryote with two genomes, one of which experiences extensive genome remodeling during development. Each round of conjugation initiates a cascade of events that construct a transcriptionally active somatic genome from a scrambled germline genome, with considerable help from both long and small noncoding RNAs. This process of genome remodeling entails massive DNA deletion and reshuffling of remaining DNA segments to form functional genes from their interrupted and scrambled germline precursors. The use of Oxytricha as a model system provides an opportunity to study an exaggerated form of programmed genome rearrangement. Furthermore, studying the mechanisms that maintain nuclear dimorphism and mediate genome rearrangement has demonstrated a surprising plasticity and diversity of noncoding RNA pathways, with new roles that go beyond conventional gene silencing. Another aspect of ciliate genetics is their unorthodox patterns of RNA-mediated, epigenetic inheritance that rival Mendelian inheritance. This review takes the reader through the key experiments in a model eukaryote that led to fundamental discoveries in RNA biology and pushes the biological limits of DNA processing.

The morphology and infraciliature of two hypotrichous ciliates, Oxytricha paragranulifera n. sp. and Oxytricha granulifera Foissner and Adam, 1983, collected respectively from the surface of a sandy soil in the Huguang mangrove forest, Zhanjiang, China, and the surface of soil in a forest beside Ziwu Road, Xian, north-west China, were examined. O. paragranulifera n. sp. is characterized by an elongate body with slightly tapered anterior end, two macronuclear nodules and two micronuclei, paroral and endoral in Stylonychia-pattern, colourless cortical granules distributed in clusters or irregular short rows, adoral zone occupying 37 % of the body length, marginal rows almost confluent posteriorly, six dorsal kineties and three caudal cirri, caudal cirri and dorsal bristles almost indistinguishable when viewed in vivo. The well-known O. granulifera Foissner and Adam, 1983 was also redescribed and can be separated from the novel species by having cortical granules arranged along dorsal kineties and marginal rows on both sides (vs grouped in clusters as well as in short irregular rows), paroral and endoral in Oxytricha-pattern (vs in Stylonychia-pattern), macronuclear nodules obviously detached (vs adjacent) and a non-saline terrestrial habitat (vs saline terrestrial). The separation of these two taxa is also firmly supported by the molecular data, which show a significant difference between the two in their SSU rRNA gene sequences (similarity 97.1 %). Phylogenetic analyses based on SSU rRNA gene sequence data suggest a close relationship within the Oxytrichidae assemblage between O. paragranulifera n. sp. and O. granulifera., (© 2014 IUMS.)

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The RNA polymerase II (Pol-II) holoenzyme, responsible for messenger RNA production, typically consists of 10-12 subunits. Our laboratory previously demonstrated that maternally deposited, long, noncoding, template RNAs are essential for programmed genome rearrangements in the ciliate Oxytricha trifallax. Here we show that such RNAs are bidirectionally transcribed and transported to the zygotic nucleus. The gene encoding the second-largest subunit of Pol-II, Rpb2, has undergone gene duplication, and the two paralogs, Rpb2-a and -b, display different expression patterns. Immunoprecipitation of double-stranded RNAs identified an association with Rpb2-a. Through immunoprecipitation and mass spectrometry, we show that Rpb2-a in early zygotes appears surprisingly unassociated with other Pol II subunits. A partial loss of function of Rpb2-a leads to an increase in expression of transposons and other germline-limited satellite repeats. We propose that evolutionary divergence of the Rpb2 paralogs has led to acquisition of transcription-independent functions during sexual reproduction that may contribute to the negative regulation of germline gene expression., (Copyright © 2014 by the Genetics Society of America.)

Methylation of cytosine DNA residues is a well-studied epigenetic modification with important roles in formation of heterochromatic regions of the genome, and also in tissue-specific repression of transcription. However, we recently found that the ciliate Oxytricha uses methylcytosine in a novel DNA elimination pathway important for programmed genome restructuring. Remarkably, mounting evidence suggests that methylcytosine can play a dual role in ciliates, repressing gene expression during some life-stages and directing DNA elimination in others. In this essay, I describe these recent advances in the DNA methylation field and discuss whether this unexpected novel role for methylcytosine in DNA elimination might be more widely conserved in eukaryotic biology, particularly in apoptotic pathways., (© 2014 WILEY Periodicals, Inc.)

The somatic nucleus of Oxytricha trifallax contains over 15,000 different chromosomes, most containing a single gene. Analysis of this 50 Mb genome uncovers novel regulatory strategies and adaptive potential when gene copy number and allelic frequency are no longer constrained by genetic linkage., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We introduce ciliated protozoa, and more specifically the stichotrichous ciliates Oxytricha and Stylonychia, as biological model systems for the analysis of programmed DNA-reorganization processes during nuclear differentiation. These include DNA excision, DNA elimination, reordering of gene segments and specific gene amplification. We show that small nuclear RNAs specify DNA sequences to be excised or retained, but also discuss the need for a RNA template molecule derived from the parental nucleus for these processes. This RNA template guides reordering of gene segments to become functional genes and determines gene copy number in the differentiated nucleus. Since the template is derived from the parental macronucleus, gene reordering and DNA amplification are inherited in a non-Mendelian epigenetic manner.

Competing Interests: The author has declared that no competing interests exist.

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor "silent" germline micronuclear genome by a process of "unscrambling" and fragmentation. The tiny macronuclear "nanochromosomes" typically encode single, protein-coding genes (a small portion, 10%, encode 2-8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease., Competing Interests: The authors have declared that no competing interests exist.

Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling., Competing Interests: The authors have declared that no competing interests exist.

Genome duality in ciliated protozoa offers a unique system to showcase their epigenome as a model of inheritance. In Oxytricha, the somatic genome is responsible for vegetative growth, whereas the germline contributes DNA to the next sexual generation. Somatic nuclear development removes all transposons and other so-called "junk" DNA, which comprise ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally deleted regions leads to their retention in later generations. Our findings highlight small RNAs as powerful transgenerational carriers of epigenetic information for genome programming., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Studies describing intricate patterns of DNA methylation in nematode and ciliate are controversial due to the uncertainty of genomic evolutionary conservation of DNA methylation enzymes.

Background: Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells., Results: Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications occur only during nuclear development and programmed genome rearrangement. We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing. We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce demethylation of both somatic and germline sequence elements during genome rearrangements, with consequent elevated levels of germline-limited repetitive elements in exconjugant cells., Conclusions: These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we propose that this motif recruits DNA modification machinery to specific chromosomes in the parental macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination during genome rearrangements.

Ciliated protozoa are peculiar for their nuclear dimorphism, wherein two types of nuclei divide nuclear functions: a germline micronucleus (MIC) is transcriptionally inert during vegetative growth, but serves as the genetic blueprint for the somatic macronucleus (MAC), which is responsible for all transcripts supporting cell growth and reproduction. While all the advantages/disadvantages associated with nuclear dimorphism are not clear, an essential advantage seems to be the ability to produce a highly polyploid MAC, which then allows for the maintenance of extremely large single cells - many ciliate cells are larger than small metazoa. In some ciliate classes, chromosomes in the MAC are extensively fragmented to create extremely short chromosomes that often carry single genes, and these chromosomes may be present in different copy numbers, resulting in different ploidies. While using gene copy number to regulate gene expression is limited in most eukaryotic systems, the extensive fragmentation in some ciliate classes provides this opportunity to every MAC gene. However, it is still unclear if this mechanism is in fact used extensively in these ciliates. To address this, we have quantified copy numbers of 11 MAC chromosomes and their gene expression in Oxytricha trifallax (CI: Spirotrichea). We compared copy numbers between two subpopulations of O. trifallax, and copy numbers of 7 orthologous genes between O. trifallax and the closely related Stylonychia lemnae. We show that copy numbers of MAC chromosomes are variable, dynamic, and positively correlated to gene expression. These features might be conserved in all spirotrichs, and might exist in other classes of ciliates with heavily fragmented MAC chromosomes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Several independent lines of evidence suggest that the modern genetic system was preceded by the 'RNA world' in which RNA genes encoded RNA catalysts. Current gaps in our conceptual framework of early genetic systems make it difficult to imagine how a stable RNA genome may have functioned and how the transition to a DNA genome could have taken place. Here we use the single-celled ciliate, Oxytricha, as an analog to some of the genetic and genomic traits that may have been present in organisms before and during the establishment of a DNA genome. Oxytricha and its close relatives have a unique genome architecture involving two differentiated nuclei, one of which encodes the genome on small, linear nanochromosomes. While its unique genomic characteristics are relatively modern, some physiological processes related to the genomes and nuclei of Oxytricha may exemplify primitive states of the developing genetic system., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Oxytricha trifallax - an established model organism for studying genome rearrangements, chromosome structure, scrambled genes, RNA-mediated epigenetic inheritance, and other phenomena - has been the subject of a nomenclature controversy for several years. Originally isolated as a sibling species of O. fallax, O. trifallax was reclassified in 1999 as Sterkiella histriomuscorum, a previously identified species, based on morphological similarity. The proper identification of O. trifallax is crucial to resolve in order to prevent confusion in both the comparative genomics and the general scientific communities. We analyzed nine conserved nuclear gene sequences between the two given species and several related ciliates. Phylogenetic analyses suggest that O. trifallax and a bona fide S. histriomuscorum have accumulated significant evolutionary divergence from each other relative to other ciliates such that they should be unequivocally classified as separate species. We also describe the original isolation of O. trifallax, including its comparison to O. fallax, and we provide criteria to identify future isolates of O. trifallax., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28-29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2'O-methylation at their 3' ends. We propose models for the role of these "27macRNAs" in macronuclear development.

The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (~70 kb) plus a ~5-kb linear plasmid bearing mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps- 2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence. The increased size of the Oxytricha mitochondrial genome relative to other ciliates is primarily a consequence of terminal expansions, rather than the retention of ancestral mitochondrial genes. Successive segmental duplications, visible in one of the two Oxytricha mitochondrial subterminal regions, appear to have contributed to the genome expansion. Consistent with pseudogene formation and decay, the subtermini possess shorter, more loosely packed open reading frames than the remainder of the genome. The mitochondrial plasmid shares a 251-bp region with 82% identity to the mitochondrial chromosome, suggesting that it most likely integrated into the chromosome at least once. This region on the chromosome is also close to the end of the most terminal member of a series of duplications, hinting at a possible association between the plasmid and the duplications. The presence of mitochondrial telomeres on the mitochondrial plasmid suggests that such plasmids may be a vehicle for lateral transfer of telomeric sequences between mitochondrial genomes. We conjecture that the extreme divergence observed in ciliate mitochondrial genomes may be due, in part, to repeated invasions by relatively error-prone DNA polymerase-bearing mobile elements.

We characterized and compared the fluorescence properties of various well-defined G-quadruplex structures. The increase of intrinsic fluorescence of G-rich DNA sequences when they form G-quadruplexes can be used to monitor the folding and unfolding of G-quadruplexes as a function of cations and temperature. The temperature-dependent fluorescence spectra of different G-quadruplexes also exhibit characteristic patterns. Thus, the stability and possibly also the structure of G-quadruplexes can be characterized and distinguished by their intrinsic fluorescence spectra., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We took advantage of the unusual genomic organization of the ciliate Oxytricha trifallax to screen for eukaryotic non-coding RNA (ncRNA) genes. Ciliates have two types of nuclei: a germ line micronucleus that is usually transcriptionally inactive, and a somatic macronucleus that contains a reduced, fragmented and rearranged genome that expresses all genes required for growth and asexual reproduction. In some ciliates including Oxytricha, the macronuclear genome is particularly extreme, consisting of thousands of tiny 'nanochromosomes', each of which usually contains only a single gene. Because the organism itself identifies and isolates most of its genes on single-gene nanochromosomes, nanochromosome structure could facilitate the discovery of unusual genes or gene classes, such as ncRNA genes. Using a draft Oxytricha genome assembly and a custom-written protein-coding genefinding program, we identified a subset of nanochromosomes that lack any detectable protein-coding gene, thereby strongly enriching for nanochromosomes that carry ncRNA genes. We found only a small proportion of non-coding nanochromosomes, suggesting that Oxytricha has few independent ncRNA genes besides homologs of already known RNAs. Other than new members of known ncRNA classes including C/D and H/ACA snoRNAs, our screen identified one new family of small RNA genes, named the Arisong RNAs, which share some of the features of small nuclear RNAs.

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNA-mediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha's somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells.

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ∼ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.

Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]₁₀ repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]₄ and [TTTTGGGG]₄ are thermally more stable compared to human [TTAGGG]₄. Consistent with this, replication of vectors with ciliate [TTGGGG]₁₀ repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]₁₀ vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. Although several nucleoprotein complexes have been described at the telomeres of different organisms, it is still unclear how they confer a structural identity to chromosome ends in order to mask them from DNA repair and to ensure their proper replication. In this review, we describe how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guaranty the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We propose that telomeric nucleoprotein complexes orient cell fate through dynamic transitions in their structures and their organization., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)