Nejvíce citovaný článek - PubMed ID 15161972
Telomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. Expression of the reverse-transcriptase telomerase allows for extension of telomeric repeats to counteract natural telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly nondegenerate and that the telomeres form a protective structure, with a subset ending with a 3' overhang and another subset presenting a blunt end. Although telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that telomeres shorten progressively in mutants of this gene. Telomerase mutants eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.
- MeSH
- Chlamydomonas reinhardtii genetika MeSH
- genetická variace MeSH
- homeostáza telomer MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- repetitivní sekvence nukleových kyselin MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- telomerasa chemie genetika metabolismus MeSH
- telomery genetika MeSH
- zkracování telomer MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- telomerasa 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.
- Klíčová slova
- AtPOT1a, PURα1, Pontin, Reptin, TAP-MS, Telomerase,
- MeSH
- Arabidopsis enzymologie genetika MeSH
- buněčné jádro enzymologie MeSH
- chromatografie afinitní MeSH
- exprese genu MeSH
- interakční proteinové domény a motivy MeSH
- kultivované buňky MeSH
- mapování interakce mezi proteiny MeSH
- mapy interakcí proteinů MeSH
- multimerizace proteinu MeSH
- proteiny huseníčku genetika izolace a purifikace metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- telomerasa genetika izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny huseníčku MeSH
- telomerasa MeSH
Mph1 is a member of the conserved FANCM family of DNA motor proteins that play key roles in genome maintenance processes underlying Fanconi anemia, a cancer predisposition syndrome in humans. Here, we identify Mte1 as a novel interactor of the Mph1 helicase in Saccharomyces cerevisiae. In vitro, Mte1 (Mph1-associated telomere maintenance protein 1) binds directly to DNA with a preference for branched molecules such as D loops and fork structures. In addition, Mte1 stimulates the helicase and fork regression activities of Mph1 while inhibiting the ability of Mph1 to dissociate recombination intermediates. Deletion of MTE1 reduces crossover recombination and suppresses the sensitivity of mph1Δ mutant cells to replication stress. Mph1 and Mte1 interdependently colocalize at DNA damage-induced foci and dysfunctional telomeres, and MTE1 deletion results in elongated telomeres. Taken together, our data indicate that Mte1 plays a role in regulation of crossover recombination, response to replication stress, and telomere maintenance.
- Klíčová slova
- DNA repair, Mph1, Mte1, genome integrity, homologous recombination, telomere maintenance,
- MeSH
- crossing over (genetika) genetika MeSH
- DEAD-box RNA-helikasy genetika metabolismus MeSH
- delece genu MeSH
- fyziologický stres genetika MeSH
- homeostáza telomer genetika MeSH
- proteiny vázající telomery genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DEAD-box RNA-helikasy MeSH
- MPH1 protein, S cerevisiae MeSH Prohlížeč
- Mte1 protein, S cerevisiae MeSH Prohlížeč
- proteiny vázající telomery MeSH
- Saccharomyces cerevisiae - proteiny 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.
Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.
- Klíčová slova
- Arabidopsis, QTL, centromere-mediated genome elimination, haploid, telomere,
- MeSH
- Arabidopsis genetika MeSH
- genetická variace * MeSH
- jednonukleotidový polymorfismus MeSH
- lokus kvantitativního znaku MeSH
- mapování chromozomů MeSH
- molekulární evoluce MeSH
- populační genetika MeSH
- selekce (genetika) * MeSH
- telomery * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
