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

To elucidate the molecular nature of evolutionary changes of telomeres in the plant order Asparagales, we aimed to characterize telomerase RNA subunits (TRs) in these plants. The unusually long telomere repeat unit in Allium plants (12 nt) allowed us to identify TRs in transcriptomic data of representative species of the Allium genus. Orthologous TRs were then identified in Asparagales plants harbouring telomere DNA composed of TTAGGG (human type) or TTTAGGG (Arabidopsis-type) repeats. Further, we identified TRs across the land plant phylogeny, including common model plants, crop plants, and plants with unusual telomeres. Several lines of functional testing demonstrate the templating telomerase function of the identified TRs and disprove a functionality of the only previously reported plant telomerase RNA in Arabidopsis thaliana. Importantly, our results change the existing paradigm in plant telomere biology which has been based on the existence of a relatively conserved telomerase reverse transcriptase subunit (TERT) associating with highly divergent TRs even between closely related plant taxa. The finding of a monophyletic origin of genuine TRs across land plants opens the possibility to identify TRs directly in transcriptomic or genomic data and/or predict telomere sequences synthesized according to the respective TR template region.

• MeSH
• Allium genetics   MeSH
• Arabidopsis genetics   MeSH
• Asparagales genetics   MeSH
• Phylogeny * MeSH
• Genome, Plant genetics   MeSH
• Humans MeSH
• Evolution, Molecular * MeSH
• RNA genetics   MeSH
• Telomerase genetics   MeSH
• Telomere genetics   MeSH
• Embryophyta genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA MeSH
• Telomerase MeSH
• telomerase RNA MeSH Browser

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

In tobacco, three sequence variants of the TERT gene have been described. We revealed unbalanced levels of TERT variant transcripts in vegetative tobacco tissues and enhanced TERT transcription and telomerase activity in reproductive tissues. Telomerase is a ribonucleoprotein complex responsible for the maintenance of telomeres, structures delimiting ends of linear eukaryotic chromosomes. In the Nicotiana tabacum (tobacco) allotetraploid plant, three sequence variants (paralogs) of the gene coding for the telomerase reverse transcriptase subunit (TERT) have been described, two of them derived from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one originated from the N. tomentosiformis paternal genome (TERT_Ct). In this work, we analyzed the transcription of TERT variants in correlation with telomerase activity in tobacco tissues. High and approximately comparable levels of TERT_Ct and TERT_Cs transcripts were detected in seedlings, roots, flower buds and leaves, while the transcript of the TERT_D variant was markedly underrepresented. Similarly, in N. sylvestris tissues, TERT_Cs transcript significantly predominated. A specific pattern of TERT transcripts was found in samples of tobacco pollen with the TERT_Cs variant clearly dominating particularly at the early stage of pollen development. Detailed analysis of TERT_C variants representation in functionally distinct fractions of pollen transcriptome revealed their prevalence in large ribonucleoprotein particles encompassing translationally silent mRNA; only a minority of TERT_Ct and TERT_Cs transcripts were localized in actively translated polysomes. Histones of the TERT_C chromatin were decorated predominantly with the euchromatin-specific epigenetic modification in both telomerase-positive and telomerase-negative tobacco tissues. We conclude that the existence and transcription pattern of tobacco TERT paralogs represents an interesting phenomenon and our results indicate its functional significance. Nicotiana species have again proved to be appropriate and useful model plants in telomere biology studies.

• Keywords
• Gene sequence variant, Pollen, Polyploids, Telomerase, Telomere, Transcription,
• MeSH
• Cell Nucleus genetics   MeSH
• Chromatin Immunoprecipitation MeSH
• Euchromatin metabolism   MeSH
• Transcription, Genetic MeSH
• Genetic Variation * MeSH
• Histones metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Organ Specificity genetics   MeSH
• Polyribosomes metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Pollen Tube growth & development   MeSH
• Gene Expression Regulation, Plant * MeSH
• Nicotiana genetics   MeSH
• Telomerase genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Euchromatin MeSH
• Histones MeSH
• RNA, Messenger 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

Telomeres are nucleoprotein structures that distinguish native chromosomal ends from double-stranded breaks. They are maintained by telomerase that adds short G-rich telomeric repeats at chromosomal ends in most eukaryotes and determines the TnAmGo sequence of canonical telomeres. We employed an experimental approach that was based on detection of repeats added by telomerase to identify the telomere sequence type forming the very ends of chromosomes. Our previous studies that focused on the algal order Chlamydomonadales revealed several changes in telomere motifs that were consistent with the phylogeny and supported the concept of the Arabidopsis-type sequence being the ancestral telomeric motif for green algae. In addition to previously described independent transitions to the Chlamydomonas-type sequence, we report that the ancestral telomeric motif was replaced by the human-type sequence in the majority of algal species grouped within a higher order clade, Caudivolvoxa. The Arabidopsis-type sequence was apparently retained in the Polytominia clade. Regarding the telomere sequence, the Chlorogonia clade within Caudivolvoxa bifurcates into two groups, one with the human-type sequence and the other group with the Arabidopsis-type sequence that is solely formed by the Chlorogonium species. This suggests that reversion to the Arabidopsis-type telomeric motif occurred in the common ancestral Chlorogonium species. The human-type sequence is also synthesized by telomerases of algal strains from Arenicolinia, Dunaliellinia and Stephanosphaerinia, except a distinct subclade within Stephanosphaerinia, where telomerase activity was not detected and a change to an unidentified telomeric motif might arise. We discuss plausible reasons why changes in telomeric motifs were tolerated during evolution of green algae.

• Keywords
• 18S rDNA phylogeny, Green algae, TRAP, Telomerase activity, Telomere evolution,
• MeSH
• Amino Acid Motifs genetics   MeSH
• Phylogeny MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Telomerase genetics   MeSH
• Telomere genetics   MeSH
• Volvocida genetics   MeSH
• Telomere Shortening genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH
• RNA, Ribosomal, 18S MeSH
• Telomerase 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

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.

• Keywords
• 45S rDNA, Arabidopsis thaliana, DNA repair, FAS1, FAS2, RAD51B, chromatin assembly factor 1, genome instability,
• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Chromatin Assembly Factor-1 genetics   metabolism   MeSH
• Genomic Instability genetics   physiology   MeSH
• DNA Repair genetics   physiology   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• DNA, Ribosomal genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin Assembly Factor-1 MeSH
• FAS protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• RAD51B protein, Arabidopsis MeSH Browser
• DNA, Ribosomal MeSH

Dysfunction of chromatin assembly factor 1 in FASCIATA mutants (fas) of Arabidopsis thaliana results in progressive loss of telomeric DNA. Although replicative telomere shortening is typically associated with incomplete resynthesis of their ends by telomerase, no change in telomerase activity could be detected in vitro in extracts from fas mutants. Besides a possible telomerase malfunction, the telomere shortening in fas mutants could presumably be due to problems with conventional replication of telomeres. To distinguish between the possible contribution of suboptimal function of telomerase in fas mutants under in vivo conditions and problems in conventional telomere replication, we crossed fas and tert (telomerase reverse transcriptase) knockout mutants and analyzed telomere shortening in segregated fas mutants, tert mutants, and double fas tert mutants in parallel. We demonstrate that fas tert knockouts show greater replicative telomere shortening than that observed even in the complete absence of telomerase (tert mutants). While the effect of tert and fas mutations on telomere lengths in double mutants is additive, manifestations of telomere dysfunction in double fas tert mutants (frequency of anaphase bridges, onset of chromosome end fusions, and common involvement of 45S rDNA in chromosome fusion sites) are similar to those in tert mutants. We conclude that in addition to possible impairment of telomerase action, a further mechanism contributes to telomere shortening in fas mutants.

• MeSH
• Arabidopsis enzymology   genetics   metabolism   MeSH
• Chromosomes, Plant genetics   metabolism   MeSH
• Chromatin Assembly Factor-1 genetics   metabolism   MeSH
• Mutation * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• RNA Splicing Factors MeSH
• Telomerase genetics   metabolism   MeSH
• Telomere genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• At2g20020 protein, Arabidopsis MeSH Browser
• Chromatin Assembly Factor-1 MeSH
• FAS protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• RNA Splicing Factors MeSH
• Telomerase MeSH
• TERT protein, Arabidopsis MeSH Browser

Telomerase, an enzyme responsible for the maintenance of linear chromosome ends, is precisely regulated during plant development. In animals, involvement of the epigenetic state of the telomerase reverse transcriptase (TERT) gene in the complex regulation of telomerase activity has been reported. To reveal whether epigenetic mechanisms participate in the regulation of plant telomerase, the relationship between telomerase activity in tissues of Arabidopsis thaliana and DNA methylation and histone modifications in the A. thaliana TERT (AtTERT) upstream region was studied. As expected, a gradual decrease of telomerase activity during leaf maturation was observed. A different pattern with a more progressive loss of telomerase activity and AtTERT transcription during leaf development was revealed in MET1 gene-knockout mutants. Analysis of DNA methylation in the AtTERT upstream region showed low levels of methylated cytosines without notable differences between telomerase-positive and telomerase-negative wild-type tissues. Surprisingly, a high level of CG methylation was found in the AtTERT coding region, although this type of methylation is a characteristic attribute of constitutively expressed genes. Analysis of chromatin modifications in the AtTERT upstream region and in exon 5 showed increased loading of the H3K27me3 mark in the telomerase-negative mature leaf compared to telomerase-positive seedlings, whereas H3K4me3, H3K9Ac, and H3K9me2 were approximately at the same level. Consistently, the chromatin structure of the AtTERT gene was maintained. These results are discussed in the context of the general involvement of epigenetic mechanisms in the regulation of gene expression and with respect to similar studies performed in animal models.

• MeSH
• Arabidopsis enzymology   genetics   growth & development   metabolism   MeSH
• Epigenesis, Genetic MeSH
• Euchromatin metabolism   MeSH
• Exons MeSH
• Histones metabolism   MeSH
• Methylation MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Telomerase genetics   metabolism   MeSH
• Gene Silencing * MeSH
• Up-Regulation MeSH
• Gene Expression Regulation, Developmental MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Euchromatin MeSH
• Histones MeSH
• Arabidopsis Proteins MeSH
• Telomerase MeSH