RESUMO
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.
Assuntos
Arabidopsis/genética , Telomerase/química , Telomerase/genética , Telomerase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico/genética , Nucléolo Celular/enzimologia , Nucléolo Celular/genética , Núcleo Celular/enzimologia , Núcleo Celular/genética , Regulação da Expressão Gênica de Plantas , Sinais de Localização Nuclear/genética , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Splicing de RNA , Relação Estrutura-Atividade , Nicotiana/genéticaRESUMO
Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability.
Assuntos
Adenina/análogos & derivados , Citidina/análogos & derivados , DNA de Plantas/efeitos dos fármacos , Nicotiana/efeitos dos fármacos , Telomerase/metabolismo , Telômero/efeitos dos fármacos , Adenina/farmacologia , Células Cultivadas , Citidina/farmacologia , Metilação de DNA/efeitos dos fármacos , DNA de Plantas/química , Ativação Enzimática/efeitos dos fármacos , Epigênese Genética , Nucleossomos/efeitos dos fármacos , Nucleossomos/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Telômero/química , Telômero/metabolismo , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Telomeres are nucleoprotein structures ensuring the stability of eukaryotic chromosome ends. Two protein families, TRFL (TFL-Like) and SMH (Single-Myb-Histone), containing a specific telobox motif in their Myb domain, have been identified as potential candidates involved in a functional nucleoprotein structure analogous to human "shelterin" at plant telomeres. We analyze the DNA-protein interaction of the full-length and truncated variants of AtTRB1, a SMH-family member with a typical structure: N-terminal Myb domain, central H1/5 domain and C-terminal coiled-coil. We show that preferential interaction of AtTRB1 with double-stranded telomeric DNA is mediated by the Myb domain, while the H1/5 domain is involved in non-specific DNA-protein interaction and in the multimerization of AtTRB1.