RESUMEN
In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.
Asunto(s)
Himenópteros , Telomerasa , Animales , Telomerasa/genética , Telomerasa/metabolismo , Himenópteros/genética , Filogenia , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Conformación de Ácido Nucleico , ARN/genética , Plantas/genética , Eucariontes/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Telomerasa , Telomerasa/genética , Telomerasa/metabolismo , Telomerasa/química , Arabidopsis/genética , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , ARN/metabolismo , ARN/genética , Técnicas del Sistema de Dos Híbridos , ARN de Planta/genética , ARN de Planta/metabolismo , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Adenosina Trifosfatasas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Mutación/genéticaRESUMEN
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.
Asunto(s)
Evolución Molecular , Filogenia , ARN/genética , Telomerasa/genética , Telómero/genética , Allium/genética , Arabidopsis/genética , Asparagales/genética , Embryophyta/genética , Genoma de Planta/genética , HumanosRESUMEN
The gene coding for the telomerase reverse transcriptase (TERT) is essential for the maintenance of telomeres. Previously we described the presence of three TERT paralogs in the allotetraploid plant Nicotiana tabacum, while a single TERT copy was identified in the paleopolyploid model plant Arabidopsis thaliana. Here we examine the presence, origin and functional status of TERT variants in allotetraploid Nicotiana species of diverse evolutionary ages and their parental genome donors, as well as in other diploid and polyploid plant species. A combination of experimental and in silico bottom-up analyses of TERT gene copies in Nicotiana polyploids revealed various patterns of retention or loss of parental TERT variants and divergence in their functions. RT-qPCR results confirmed the expression of all the identified TERT variants. In representative plant and green algal genomes, our synteny analyses show that their TERT genes were located in a conserved locus that became advantageous after the divergence of eudicots, and the gene was later translocated in several plant groups. In various diploid and polyploid species, translocation of TERT became fixed in target loci that show ancient synapomorphy.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Dosificación de Gen , Nicotiana , Poliploidía , Telomerasa , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
ALBA DNA/RNA-binding proteins form an ancient family, which in eukaryotes diversified into two Rpp25-like and Rpp20-like subfamilies. In most studied model organisms, their function remains unclear, but they are usually associated with RNA metabolism, mRNA translatability and stress response. In plants, the enriched number of ALBA family members remains poorly understood. Here, we studied ALBA dynamics during reproductive development in Arabidopsis at the levels of gene expression and protein localization, both under standard conditions and following heat stress. In generative tissues, ALBA proteins showed the strongest signal in mature pollen where they localized predominantly in cytoplasmic foci, particularly in regions surrounding the vegetative nucleus and sperm cells. Finally, we demonstrated the involvement of two Rpp25-like subfamily members ALBA4 and ALBA6 in RNA metabolism in mature pollen supported by their co-localization with poly(A)-binding protein 3 (PABP3). Collectively, we demonstrated the engagement of ALBA proteins in male reproductive development and the heat stress response, highlighting the involvement of ALBA4 and ALBA6 in RNA metabolism, storage and/or translational control in pollen upon heat stress. Such dynamic re-localization of ALBA proteins in a controlled, developmentally and environmentally regulated manner, likely reflects not only their redundancy but also their possible functional diversification in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Polen/embriología , Proteínas de Unión al ARN/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/genética , Respuesta al Choque Térmico/fisiología , Microscopía Confocal , Proteínas de Unión a Poli(A)/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas de Unión al ARN/genética , Estrés Fisiológico/genéticaRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Telomerasa/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ensamble y Desensamble de Cromatina , Replicación del ADN , Regulación de la Expresión Génica de las Plantas , Aparato de Golgi/metabolismo , Mitocondrias/metabolismo , Complejos Multiproteicos/metabolismo , Nucleosomas/metabolismo , Péptidos/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas , Procesamiento Postranscripcional del ARN/genética , Ribosomas/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/metabolismo , Transcripción GenéticaRESUMEN
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/ß-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.
Asunto(s)
Arabidopsis/enzimología , Proteínas del Dominio Armadillo/metabolismo , Telomerasa/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas del Dominio Armadillo/genética , Genes Reporteros , Holoenzimas , Humanos , Telomerasa/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Phylogenetic divergence in Asparagales plants is associated with switches in telomere sequences. The last switch occurred with divergence of the genus Allium (Amaryllidaceae) from the other Allioideae (formerly Alliaceae) genera, resulting in uncharacterized telomeres maintained by an unknown mechanism. To characterize the unknown Allium telomeres, we applied a combination of bioinformatic processing of transcriptomic and genomic data with standard approaches in telomere biology such as BAL31 sensitivity tests, terminal restriction fragment analysis, the telomere repeat amplification protocol (TRAP), and fluorescence in situ hybridization (FISH). Using these methods, we characterize the unusual telomeric sequence (CTCGGTTATGGG)n present in Allium species, demonstrate its synthesis by telomerase, and characterize the telomerase reverse transcriptase (TERT) subunit of Allium cepa. Our findings open up the possibility of studying the molecular details of the evolutionary genetic change in Allium telomeres and its possible role in speciation. Experimental studies addressing the implications of this change in terms of the interplay of telomere components may now be designed to shed more light on telomere functions and evolution in general.
Asunto(s)
Allium/genética , Cromosomas de las Plantas/genética , Evolución Molecular , Telomerasa/metabolismo , Telómero/genética , Allium/enzimología , Secuencia de Bases , Biología Computacional , Genómica , Hibridación Fluorescente in Situ , Filogenia , Análisis de Secuencia de ADN , Telomerasa/genética , TranscriptomaRESUMEN
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.
Asunto(s)
Secuencias de Aminoácidos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Telomerasa/genética , Telómero/genética , Volvocida/genética , Secuencia de Bases , ADN Ribosómico/genética , Filogenia , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN , Acortamiento del Telómero/genéticaRESUMEN
MAIN CONCLUSION: 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.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Variación Genética , Nicotiana/genética , Especificidad de Órganos/genética , Telomerasa/genética , Núcleo Celular/genética , Inmunoprecipitación de Cromatina , Eucromatina/metabolismo , Histonas/metabolismo , Tubo Polínico/crecimiento & desarrollo , Polirribosomas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Telomerasa/metabolismo , Transcripción GenéticaRESUMEN
The characterization of unusual telomere sequence sheds light on patterns of telomere evolution, maintenance and function. Plant species from the closely related genera Cestrum, Vestia and Sessea (family Solanaceae) lack known plant telomeric sequences. Here we characterize the telomere of Cestrum elegans, work that was a challenge because of its large genome size and few chromosomes (1C 9.76 pg; n = 8). We developed an approach that combines BAL31 digestion, which digests DNA from the ends and chromosome breaks, with next-generation sequencing (NGS), to generate data analysed in RepeatExplorer, designed for de novo repeats identification and quantification. We identify an unique repeat motif (TTTTTTAGGG)n in C. elegans, occurring in ca. 30 400 copies per haploid genome, averaging ca. 1900 copies per telomere, and synthesized by telomerase. We demonstrate that the motif is synthesized by telomerase. The occurrence of an unusual eukaryote (TTTTTTAGGG)n telomeric motif in C. elegans represents a switch in motif from the 'typical' angiosperm telomere (TTTAGGG)n . That switch may have happened with the divergence of Cestrum, Sessea and Vestia. The shift in motif when it arose would have had profound effects on telomere activity. Thus our finding provides a unique handle to study how telomerase and telomeres responded to genetic change, studies that will shed more light on telomere function.
Asunto(s)
Cestrum/genética , Cromosomas de las Plantas/genética , Telómero/química , Telómero/genéticaRESUMEN
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.
Asunto(s)
Bryopsida/genética , Cromosomas de las Plantas/genética , Reparación del ADN , Telomerasa/genética , Homeostasis del Telómero/genética , Telómero/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Secuencia de Bases , Bryopsida/metabolismo , Roturas del ADN de Doble Cadena , ADN de Plantas/genética , Recombinación Homóloga , Datos de Secuencia Molecular , Mutación , Fenotipo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Telomerasa/metabolismoRESUMEN
Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein- or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ~2 × 10(5) cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.
Asunto(s)
Agrobacterium tumefaciens/genética , Proteínas de Arabidopsis/genética , ADN Bacteriano/genética , Biblioteca de Genes , Unión ProteicaRESUMEN
An experimental design was set up to understand the influence of five process parameters on the kinetics of struvite precipitation in synthetic swine wastewaters. The responses studied were the kinetics of phosphorus (P) removal, the struvite precipitation rate and the dissolution rate of amorphous calcium phosphates (ACP). The kinetic study showed that the P-removal was complete in less than 1 h and was influenced positively by the added MgO. The precipitation of struvite with MgO was confirmed to follow a first-order kinetic. This study showed that ACP co-precipitated with struvite during the first 30 min. Afterwards, ACP dissolved to maintain the phosphates balance limiting the struvite growth. An initial Mg:Ca > 1.5 induced a complete dissolution of ACP in 1 h. Another experiment was conducted and it validated the results of the statistical model. This experiment also determined that 7-10 h was the best time to recover large crystals. After 10 h, the crystals were broken by stirring.
Asunto(s)
Compuestos de Magnesio/química , Fosfatos/química , Aguas Residuales/química , Animales , Reactores Biológicos , Precipitación Química , Concentración de Iones de Hidrógeno , Cinética , Óxido de Magnesio , Modelos Estadísticos , Estruvita , Porcinos , Administración de ResiduosRESUMEN
Armadillo repeat-containing proteins (ARMCs) are a large family found throughout eukaryotes, which play prominent roles in cell adhesion, signaling and cytoskeletal regulation. The ARMC6 protein is highly conserved in primates, including humans, but to date does not have a clear function beyond initial hints of a link to cancer and telomerase activity. We report here in vitro experiments showing ARMC6 binding to DNA promoter sequences from several cancer-related genes (e.g., EGFR, VEGF and c-MYC), and also to the telomeric RNA repeat (TERRA). ARMC6 binding activity appears to recognize G-quadruplex motifs, which are being increasingly implicated as structure-based protein binding sites in chromosome maintenance and repair. In vivo investigation of ARMC6 function revealed that when this protein is overexpressed in human cell lines, there is different expression of genes connected with oncogenic pathways and those implicated in downstream non-canonical telomerase pathways (e.g., VEGF, hTERT, c-MYC, ESM1, MMP3). ARMC6 is already known to interact with human shelterin protein TRF2 and telomerase. The protein binds G-quadruplex structures and does so preferentially to RNA over DNA. As such, this protein may be an example of how a non-canonical nucleic acid structural motif allows mediation between gene regulation and telomeric chromatin rearrangement pathways.
Asunto(s)
Proteínas del Dominio Armadillo , G-Cuádruplex , Regiones Promotoras Genéticas , Telómero , Humanos , Proteínas del Dominio Armadillo/metabolismo , Proteínas del Dominio Armadillo/genética , Sitios de Unión , Línea Celular Tumoral , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , ARN/metabolismo , ARN/genética , Telomerasa/metabolismo , Telomerasa/genética , Telómero/metabolismo , Factores de TranscripciónRESUMEN
Telomere minisatellites could be present in both terminal and internal chromosomal regions. We monitored the progress of BAL-31 nuclease digestion on Arabidopsis thaliana genomic DNA prepared by standard isolation techniques to verify its cleavage at terminal and internal genomic regions. A subtelomeric position of candidate sequences was validated using conventional polymerase chain reaction (PCR), combining the C-strand-specific telomeric primer with a subtelomeric reverse primer, and confirmed by quantitative PCR (qPCR) using sequence-specific primer pairs on DNA samples after BAL-31 digestion. qPCR amplification showed a gradual decrease in subtelomeric sequence signals, in contrast to interstitial telomeric sequences from pericentromere and control sequences.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Telómero/genética , Arabidopsis/genética , Secuencia de Bases , ADN de Plantas/genéticaRESUMEN
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.
Asunto(s)
Arabidopsis/genética , Telomerasa/química , Telomerasa/genética , Telomerasa/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dominio Catalítico/genética , Nucléolo Celular/enzimología , Nucléolo Celular/genética , Núcleo Celular/enzimología , Núcleo Celular/genética , Regulación de la Expresión Génica de las Plantas , Señales de Localización Nuclear/genética , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Empalme del ARN , Relación Estructura-Actividad , Nicotiana/genéticaRESUMEN
Telomerase is essential for proper functioning of telomeres in eukaryotes. We cloned and characterised genes for the protein subunit of telomerase (TERT) in the allotetraploid Nicotiana tabacum (tobacco) and its diploid progenitor species Nicotiana sylvestris and Nicotiana tomentosiformis with the aim of determining if allopolyploidy (hybridisation and genome duplication) influences TERT activity and divergence. Two of the three sequence variants present in the tobacco genome (NtTERT-C/s and NtTERT-D) revealed similarity to two sequence variants found in N. sylvestris and another variant (NtTERT-C/t) was similar to TERT of N. tomentosiformis. Variants of N. sylvestris origin showed less similarity to each other (80.5 % in the genomic region; 90.1 % in the coding sequence) than that between the NtTERT-C/s and NtTERT-C/t variants (93.6 and 97.2 %, respectively). The NtTERT-D variant was truncated at the 5' end, and indels indicated that it was a pseudogene. All tobacco variants were transcribed and alternatively spliced sequences were detected. Analysis of gene arrangements uncovered a novel exon in the N-terminal domain of TERT variants, a feature that is likely to be commonly found in Solanaceae species. In addition, species-specific duplications were observed within exon 5. The putative function, copy number and evolutionary origin of these NtTERT sequence variants are discussed.
Asunto(s)
Nicotiana/genética , Telomerasa/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Molecular , Exones , Orden Génico , Reordenamiento Génico , Sitios Genéticos , Genoma de Planta , Intrones , Datos de Secuencia Molecular , Seudogenes , Isoformas de ARN , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Transcripción GenéticaRESUMEN
Telomeres are essential nucleoprotein structures at the very ends of linear eukaryote chromosomes. They shelter the terminal genome territories against degradation and prevent the natural chromosome ends from being recognized by repair mechanisms as double-strand DNA breaks.There are two basic characteristics of telomeric DNA, its sequence and its length. The telomere sequence is important as a "landing area" for specific telomere-binding proteins, which function as signals and moderate the interactions required for correct telomere function. While the sequence forms the proper "landing surface" of telomeric DNA, its length is similarly important. Too short or exceptionally long telomere DNA cannot perform its function properly. In this chapter, methods for the investigation of these two basic telomere DNA characteristics are described, namely, telomere motif identification and telomere length measurement.