Analysis of plants bearing a T-DNA insertion is a potent tool of modern molecular biology, providing valuable information about the function and involvement of genes in metabolic pathways. A collection of 12 Arabidopsis thaliana lines with T-DNA insertions in the gene coding for the catalytic subunit of telomerase (AtTERT) and in adjacent regions was screened for telomerase activity [telomere repeat amplification protocol (TRAP) assay], telomere length (terminal restriction fragments), and AtTERT transcription (quantitative reverse transcription-PCR). Lines with the insertion located upstream of the start codon displayed unchanged telomere stability and telomerase activity, defining a putative minimal AtTERT promoter and the presence of a regulatory element linked to increased transcription in the line SALK_048471. Lines bearing a T-DNA insertion inside the protein-coding region showed telomere shortening and lack of telomerase activity. Transcription in most of these lines was unchanged upstream of the T-DNA insertion, while it was notably decreased downstream. The expression profile varied markedly in mutant lines harbouring insertions at the 5' end of AtTERT which showed increased transcription and abolished tissue specificity. Moreover, the line FLAG_385G01 (T-DNA insertion inside intron 1) revealed the presence of a highly abundant downstream transcript with normal splicing but without active telomerase. The role of regulatory elements found along the AtTERT gene is discussed in respect to natural telomerase expression and putative intron-mediated enhancement.

• MeSH
• Arabidopsis genetika   MeSH
• DNA bakterií genetika   MeSH
• genotyp MeSH
• inzerční mutageneze MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny huseníčku genetika   MeSH
• regulace genové exprese u rostlin MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• telomerasa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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 enzymologie   genetika   růst a vývoj   metabolismus   MeSH
• epigeneze genetická MeSH
• euchromatin metabolismus   MeSH
• exony MeSH
• histony metabolismus   MeSH
• metylace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• telomerasa genetika   metabolismus   MeSH
• umlčování genů MeSH
• upregulace MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Although telomerase (EC 2.7.7.49) is important for genome stability and totipotency of plant cells, the principles of its regulation are not well understood. Therefore, we studied subcellular localization and function of the full-length and truncated variants of the catalytic subunit of Arabidopsis thaliana telomerase, AtTERT, in planta. Our results show that multiple sites in AtTERT may serve as nuclear localization signals, as all the studied individual domains of the AtTERT were targeted to the nucleus and/or the nucleolus. Although the introduced genomic or cDNA AtTERT transgenes display expression at transcript and protein levels, they are not able to fully complement the lack of telomerase functions in tert -/- mutants. The failure to reconstitute telomerase function in planta suggests a more complex telomerase regulation in plant cells than would be expected based on results of similar experiments in mammalian model systems.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• buněčné jadérko enzymologie   genetika   MeSH
• buněčné jádro enzymologie   genetika   MeSH
• geneticky modifikované rostliny MeSH
• jaderné lokalizační signály genetika   MeSH
• katalytická doména genetika   MeSH
• listy rostlin genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteosyntéza MeSH
• regulace genové exprese u rostlin MeSH
• sestřih RNA MeSH
• tabák genetika   MeSH
• telomerasa chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

KEY MESSAGE: 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.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• holoenzymy MeSH
• lidé MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny s doménou armadillo genetika   metabolismus   MeSH
• reportérové geny MeSH
• techniky dvojhybridového systému MeSH
• telomerasa genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Telomerase maturation and recruitment to telomeres is regulated by several telomerase- and telomere-associated proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively, and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase (AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus, and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genome-wide phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction of AtRuvBL genes in heterozygous T-DNA insertion A. thaliana mutants results in reduced telomerase activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• buněčné jadérko metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• fylogeneze MeSH
• jaderné proteiny MeSH
• katalytická doména MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• proteiny vázající telomery metabolismus   MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH