Most eukaryotic organisms employ a telomerase complex for the maintenance of chromosome ends. The core of this complex is composed of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) subunits. The TERT reverse transcriptase (RT) domain synthesises telomeric DNA using the TR template sequence. The other TERT domains contribute to this process in different ways. In particular, the TERT RNA-binding domain (TRBD) interacts with specific TR motif(s). Using a yeast 3-hybrid system, we show the critical role of Arabidopsis thaliana (At) TRBD and embryophyta-conserved KRxR motif in the unstructured linker preceding the TRBD domain for binding to the recently identified AtTR subunit. We also show the essential role of the predicted P4 stem and pseudoknot AtTR structures and provide evidence for the binding of AtTRBD to pseudoknot and KRxR motif stabilising interaction with the P4 stem structure. Our results thus provide the first insight into the core part of the plant telomerase complex.

• Keywords
• A.thaliana telomerase, AtTERT, AtTR, Protein-RNA interactions, Yeast three-hybrid,
• MeSH
• Arabidopsis * genetics   enzymology   MeSH
• Nucleic Acid Conformation MeSH
• Arabidopsis Proteins * genetics   metabolism   chemistry   MeSH
• RNA, Plant genetics   metabolism   MeSH
• RNA metabolism   genetics   MeSH
• Two-Hybrid System Techniques MeSH
• Telomerase * genetics   metabolism   chemistry   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH

Telomerase, an essential enzyme that maintains chromosome ends, is important for genome integrity and organism development. Various hypotheses have been proposed in human, ciliate and yeast systems to explain the coordination of telomerase holoenzyme assembly and the timing of telomerase performance at telomeres during DNA replication or repair. However, a general model is still unclear, especially pathways connecting telomerase with proposed non-telomeric functions. To strengthen our understanding of telomerase function during its intracellular life, we report on interactions of several groups of proteins with the Arabidopsis telomerase protein subunit (AtTERT) and/or a component of telomerase holoenzyme, POT1a protein. Among these are the nucleosome assembly proteins (NAP) and the minichromosome maintenance (MCM) system, which reveal new insights into the telomerase interaction network with links to telomere chromatin assembly and replication. A targeted investigation of 176 candidate proteins demonstrated numerous interactions with nucleolar, transport and ribosomal proteins, as well as molecular chaperones, shedding light on interactions during telomerase biogenesis. We further identified protein domains responsible for binding and analyzed the subcellular localization of these interactions. Moreover, additional interaction networks of NAP proteins and the DOMINO1 protein were identified. Our data support an image of functional telomerase contacts with multiprotein complexes including chromatin remodeling and cell differentiation pathways.

• Keywords
• Arabidopsis, chromatin, folding, mitochondria, protein–protein interaction, replication, telomerase, transport,
• MeSH
• Arabidopsis metabolism   MeSH
• Transcription, Genetic MeSH
• Golgi Apparatus metabolism   MeSH
• Telomere Homeostasis MeSH
• Protein Interaction Maps MeSH
• Mitochondria metabolism   MeSH
• Multiprotein Complexes metabolism   MeSH
• Nucleosomes metabolism   MeSH
• Peptides metabolism   MeSH
• RNA Processing, Post-Transcriptional genetics   MeSH
• Arabidopsis Proteins chemistry   metabolism   MeSH
• Telomere-Binding Proteins metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• DNA Replication MeSH
• Chromatin Assembly and Disassembly MeSH
• Ribosomes metabolism   MeSH
• Telomerase metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Multiprotein Complexes MeSH
• Nucleosomes MeSH
• Peptides MeSH
• Arabidopsis Proteins MeSH
• Telomere-Binding Proteins MeSH
• Telomerase MeSH

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.

• MeSH
• Arabidopsis genetics   physiology   MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• Stress, Physiological MeSH
• Genome, Plant MeSH
• Genomic Instability MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-myb genetics   metabolism   MeSH
• DNA Replication * MeSH
• Signal Transduction * MeSH
• Transcription Factors genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATM protein, Arabidopsis MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR1 protein, Arabidopsis MeSH Browser
• FAS protein, Arabidopsis MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Arabidopsis Proteins MeSH
• Proto-Oncogene Proteins c-myb MeSH
• SOG1 protein, Arabidopsis MeSH Browser
• Transcription Factors MeSH
• WEE1 protein, Arabidopsis MeSH Browser

The canonical DNA polymerases involved in the replication of the genome are unable to fully replicate the physical ends of linear chromosomes, called telomeres. Chromosomal termini thus become shortened in each cell cycle. The maintenance of telomeres requires telomerase-a specific RNA-dependent DNA polymerase enzyme complex that carries its own RNA template and adds telomeric repeats to the ends of chromosomes using a reverse transcription mechanism. Both core subunits of telomerase-its catalytic telomerase reverse transcriptase (TERT) subunit and telomerase RNA (TR) component-were identified in quick succession in Tetrahymena more than 30 years ago. Since then, both telomerase subunits have been described in various organisms including yeasts, mammals, birds, reptiles and fish. Despite the fact that telomerase activity in plants was described 25 years ago and the TERT subunit four years later, a genuine plant TR has only recently been identified by our group. In this review, we focus on the structure, composition and function of telomerases. In addition, we discuss the origin and phylogenetic divergence of this unique RNA-dependent DNA polymerase as a witness of early eukaryotic evolution. Specifically, we discuss the latest information regarding the recently discovered TR component in plants, its conservation and its structural features.

• Keywords
• evolution, plant TERT, plant TR., telomerase, telomerase RNA (TR), telomerase reverse transcriptase (TERT),
• MeSH
• Biological Evolution * MeSH
• History, 20th Century MeSH
• History, 21st Century MeSH
• Eukaryota classification   genetics   metabolism   MeSH
• Phylogeny MeSH
• Humans MeSH
• RNA physiology   MeSH
• Telomerase chemistry   physiology   MeSH
• Telomere metabolism   MeSH
• Animals MeSH
• Check Tag
• History, 20th Century MeSH
• History, 21st Century MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Historical Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA MeSH
• Telomerase MeSH
• telomerase RNA MeSH Browser

Parallel research on multiple model organisms shows that while some principles of telomere biology are conserved among all eukaryotic kingdoms, we also find some deviations that reflect different evolutionary paths and life strategies, which may have diversified after the establishment of telomerase as a primary mechanism for telomere maintenance. Much more than animals, plants have to cope with environmental stressors, including genotoxic factors, due to their sessile lifestyle. This is, in principle, made possible by an increased capacity and efficiency of the molecular systems ensuring maintenance of genome stability, as well as a higher tolerance to genome instability. Furthermore, plant ontogenesis differs from that of animals in which tissue differentiation and telomerase silencing occur during early embryonic development, and the "telomere clock" in somatic cells may act as a preventive measure against carcinogenesis. This does not happen in plants, where growth and ontogenesis occur through the serial division of apical meristems consisting of a small group of stem cells that generate a linear series of cells, which differentiate into an array of cell types that make a shoot and root. Flowers, as generative plant organs, initiate from the shoot apical meristem in mature plants which is incompatible with the human-like developmental telomere shortening. In this review, we discuss differences between human and plant telomere biology and the implications for aging, genome stability, and cell and organism survival. In particular, we provide a comprehensive comparative overview of telomere proteins acting in humans and in Arabidopsis thaliana model plant, and discuss distinct epigenetic features of telomeric chromatin in these species.

• Keywords
• Arabidopsis, aging, chromatin, epigenetics, human, review, telomerase, telomere,
• MeSH
• Chromatin metabolism   MeSH
• Epigenesis, Genetic MeSH
• Humans MeSH
• Plants metabolism   MeSH
• Cellular Senescence genetics   MeSH
• Telomerase metabolism   MeSH
• Telomere metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Chromatin MeSH
• Telomerase MeSH

Arabidopsis and human ARM protein interact with telomerase. Deregulated mRNA levels of DNA repair and ribosomal protein genes in an Arabidopsis arm mutant suggest non-telomeric ARM function. The human homolog ARMC6 interacts with hTRF2. Telomerase maintains telomeres and has proposed non-telomeric functions. We previously identified interaction of the C-terminal domain of Arabidopsis telomerase reverse transcriptase (AtTERT) with an armadillo/β-catenin-like repeat (ARM) containing protein. Here we explore protein-protein interactions of the ARM protein, AtTERT domains, POT1a, TRF-like family and SMH family proteins, and the chromatin remodeling protein CHR19 using bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H) analysis, and co-immunoprecipitation. The ARM protein interacts with both the N- and C-terminal domains of AtTERT in different cellular compartments. ARM interacts with CHR19 and TRF-like I family proteins that also bind AtTERT directly or through interaction with POT1a. The putative human ARM homolog co-precipitates telomerase activity and interacts with hTRF2 protein in vitro. Analysis of Arabidopsis arm mutants shows no obvious changes in telomere length or telomerase activity, suggesting that ARM is not essential for telomere maintenance. The observed interactions with telomerase and Myb-like domain proteins (TRF-like family I) may therefore reflect possible non-telomeric functions. Transcript levels of several DNA repair and ribosomal genes are affected in arm mutants, and ARM, likely in association with other proteins, suppressed expression of XRCC3 and RPSAA promoter constructs in luciferase reporter assays. In conclusion, ARM can participate in non-telomeric functions of telomerase, and can also perform its own telomerase-independent functions.

• Keywords
• ARMC6, Armadillo/β-catenin-like repeat, AtTERT, Homologous recombination, Protein–protein interaction, Telomerase activity,
• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Holoenzymes MeSH
• Humans MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Armadillo Domain Proteins genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Two-Hybrid System Techniques MeSH
• Telomerase genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ARMC6 protein, human MeSH Browser
• Holoenzymes MeSH
• Arabidopsis Proteins MeSH
• Armadillo Domain Proteins MeSH
• Telomerase MeSH

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.

• Keywords
• AtPOT1a, PURα1, Pontin, Reptin, TAP-MS, Telomerase,
• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Cell Nucleus enzymology   MeSH
• Chromatography, Affinity MeSH
• Gene Expression MeSH
• Protein Interaction Domains and Motifs MeSH
• Cells, Cultured MeSH
• Protein Interaction Mapping MeSH
• Protein Interaction Maps MeSH
• Protein Multimerization MeSH
• Arabidopsis Proteins genetics   isolation & purification   metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Telomerase genetics   isolation & purification   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arabidopsis Proteins MeSH
• Telomerase MeSH

Telomeres, as physical ends of linear chromosomes, are targets of a number of specific proteins, including primarily telomerase reverse transcriptase. Access of proteins to the telomere may be affected by a number of diverse factors, e.g., protein interaction partners, local DNA or chromatin structures, subcellular localization/trafficking, or simply protein modification. Knowledge of composition of the functional nucleoprotein complex of plant telomeres is only fragmentary. Moreover, the plant telomeric repeat binding proteins that were characterized recently appear to also be involved in non-telomeric processes, e.g., ribosome biogenesis. This interesting finding was not totally unexpected since non-telomeric functions of yeast or animal telomeric proteins, as well as of telomerase subunits, have been reported for almost a decade. Here we summarize known facts about the architecture of plant telomeres and compare them with the well-described composition of telomeres in other organisms.

• Keywords
• plant, shelterin, telomerase, telomere, telomeric proteins, telomeric repeat binding (TRB),
• Publication type
• Journal Article MeSH
• Review MeSH

Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

• Keywords
• BiFC, MODIFIER OF snc1, metallothionein 2A, nuclear poly(A)-binding protein, oxidation-related zinc finger 2 protein, putative nuclear DNA-binding protein G2p, telobox, telomerase,
• Publication type
• Journal Article MeSH

A comparative approach in biology is needed to assess the universality of rules governing this discipline. In plant telomere research, most of the key principles were established based on studies in only single model plant, Arabidopsis thaliana. These principles include the absence of telomere shortening during plant development and the corresponding activity of telomerase in dividing (meristem) plant cells. Here we examine these principles in Physcomitrella patens as a representative of lower plants. To follow telomerase expression, we first characterize the gene coding for the telomerase reverse transcriptase subunit PpTERT in P. patens, for which only incomplete prediction has been available so far. In protonema cultures of P. patens, growing by filament apical cell division, the proportion of apical (dividing) cells was quantified and telomere length, telomerase expression and activity were determined. Our results show telomere stability and demonstrate proportionality of telomerase activity and expression with the number of apical cells. In addition, we analyze telomere maintenance in mre11, rad50, nbs1, ku70 and lig4 mutants of P. patens and compare the impact of these mutations in double-strand-break (DSB) repair pathways with earlier observations in corresponding A. thaliana mutants. Telomere phenotypes are absent and DSB repair kinetics is not affected in P. patens mutants for DSB factors involved in non-homologous end joining (NHEJ). This is compliant with the overall dominance of homologous recombination over NHEJ pathways in the moss, contrary to the inverse situation in flowering plants.

• MeSH
• Arabidopsis genetics   MeSH
• Chromosomes, Plant genetics   MeSH
• DNA, Plant genetics   MeSH
• DNA Breaks, Double-Stranded MeSH
• Phenotype MeSH
• Phylogeny MeSH
• Telomere Homeostasis genetics   MeSH
• Homologous Recombination MeSH
• Bryopsida genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Mutation MeSH
• DNA Repair * MeSH
• Plant Proteins genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Sequence Alignment MeSH
• Telomerase genetics   metabolism   MeSH
• Telomere genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Plant Proteins MeSH
• Telomerase MeSH