RESUMO
TERRA is an RNA molecule transcribed from human subtelomeric regions toward chromosome ends potentially involved in regulation of heterochromatin stability, semiconservative replication, and telomerase inhibition, among others. TERRA contains tandem repeats of the sequence GGGUUA, with a strong tendency to fold into a four-stranded arrangement known as a parallel G-quadruplex. Here, we demonstrate by using single-molecule force spectroscopy that this potential is limited by the inherent capacity of RNA to self-associate randomly and further condense into entropically more favorable structures. We stretched RNA constructions with more than four and less than eight hexanucleotide repeats, thus unable to form several G-quadruplexes in tandem, flanked by non-G-rich overhangs of random sequence by optical tweezers on a one by one basis. We found that condensed RNA stochastically blocks G-quadruplex folding pathways with a near 20% probability, a behavior that is not found in DNA analogous molecules.
Assuntos
Quadruplex G , RNA/química , Telômero/química , Sequência de Bases , Humanos , Desnaturação de Ácido Nucleico , Pinças ÓpticasRESUMO
Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.
Assuntos
Centrômero/genética , Drosophila melanogaster/genética , Motivos de Nucleotídeos/genética , Sequências de Repetição em Tandem/genética , Animais , Pareamento de Bases , Sequência de Bases , Cromossomos de Insetos/genética , DNA Satélite/genética , DimerizaçãoRESUMO
Human centromeric alpha-satellite DNA is composed of tandem arrays of two types of 171â bp monomers; type A and type B. The differences between these types are concentrated in a 17â bp region of the monomer called the A/B box. Here, we have determined the solution structure of the C-rich strand of the two main variants of the human alpha-satellite A box. We show that, under acidic conditions, the C-rich strands of two A boxes self-recognize and form a head-to-tail dimeric i-motif stabilized by four intercalated hemi-protonated C:C(+) base pairs. Interestingly, the stack of C:C(+) base pairs is capped by T:T and Hoogsteen A:T base pairs. The two main variants of the A box adopt a similar three-dimensional structure, although the residues involved in the formation of the i-motif core are different in each case. Together with previous studies showing that the B box (known as the CENP-B box) also forms dimeric i-motif structures, our finding of this non-canonical structure in the A box shows that centromeric alpha satellites in all human chromosomes are able to form i-motifs, which consequently raises the possibility that these structures may play a role in the structural organization of the centromere.
Assuntos
Centrômero/química , Substâncias Intercalantes/química , Pareamento de Bases , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
Assuntos
Drosophila melanogaster/genética , Genoma , Animais , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Biologia Computacional , Mapeamento de Sequências Contíguas , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Cromossomos Politênicos , Mapeamento por RestriçãoRESUMO
Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral target. Here, we report the first successful application of (19)F-NMR fragment-based screening to identify chemically diverse compounds that bind to an RNA molecule such as TERRA. We have built a library of 355 fluorinated fragments, and checked their interaction with a long telomeric RNA as a target molecule. The screening resulted in the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with TERRA was confirmed by (19)F- and (1)H NMR as well as by CD melting experiments. We have also explored the selectivity of the ligands for RNA G-quadruplexes and found that some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition of this particular quadruplex topology and that different ligands may recognize specific sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting binders promising lead compounds for fragment based drug discovery.
Assuntos
Antineoplásicos/química , Quadruplex G/efeitos dos fármacos , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/química , Antineoplásicos/farmacologia , Sequência de Bases , Descoberta de Drogas , Halogenação , Humanos , Ligantes , Modelos Moleculares , Neoplasias/tratamento farmacológico , Ressonância Magnética Nuclear Biomolecular , RNA/química , Sequências Repetitivas de Ácido Nucleico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Telômero/química , Telômero/metabolismoRESUMO
We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.
Assuntos
Quadruplex G , RNA/química , Humanos , RNA/genética , Dobramento de RNA , Sequências Repetitivas de Ácido Nucleico , Telômero/genéticaRESUMO
The transition of an ancestral circular genome to multiple linear chromosomes was crucial for eukaryogenesis because it allowed rapid adaptive evolution through aneuploidy. Here, we propose that the ends of nascent linear chromosomes should have had a dual function in chromosome end protection (capping) and chromosome segregation to give rise to the "proto-telomeres." Later on, proper centromeres evolved at subtelomeric regions. We also propose that both noncanonical structures based on guanine-guanine interactions and the end-protection proteins recruited by the emergent telomeric heterochromatin have been required for telomere maintenance through evolution. We further suggest that the origin of Drosophila telomeres may be reminiscent of how the first telomeres arose.
Assuntos
Cromossomos/genética , Evolução Molecular , Quadruplex G , Telômero/genética , Animais , Centrômero/genética , Humanos , Retroelementos , Telomerase/metabolismo , Telômero/metabolismoRESUMO
We report here that C6orf89, which encodes a protein that interacts with bombesin receptor subtype-3 and accelerates cell cycle progression and wound repair in human bronchial epithelial cells (Liu et al., 2011, PLoS ONE 6: e23072), encodes one soluble and two type II membrane proteins that function as histone deacetylases (HDAC) enhancers. Soluble 34/64sp is selectively targeted to the nucleolus and is retained in nucleolar organiser regions (NORs) in mitotic cells. Nucleolar 34/64sp is integrated into the ribosomal gene transcription machinery, colocalises and coimmunoprecipitates with the Pol I transcription factor UBF, and undergoes a dramatic relocalisation to the nucleolus upon the arrest of rDNA transcription, protein synthesis and PI3K/mTORC2 signalling. Membrane 42/116mp localises to the Golgi and the midbody, and its controlled ectopic expression provokes the disruption of the Golgi cisternae and hinders the separation of daughter cells and the completion of mitosis. The latter effect is also produced by the microinjection of an affinity-purified amfion antibody. The identification of C60rf89 as a gene that encodes three distinct proteins with the capacity to enhance the activity of histone deacetylases (HDACs) in the nucleolus, the Golgi and the midbody provides new information regarding the components of the acetylome and their capacity to interact with different functional groups in the cell.
Assuntos
Núcleo Celular/metabolismo , Complexo de Golgi/metabolismo , Região Organizadora do Nucléolo/metabolismo , Proteínas/genética , Ciclo Celular/genética , Núcleo Celular/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Complexo de Golgi/genética , Células HeLa , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Mitose/genética , Região Organizadora do Nucléolo/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas/metabolismo , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismoRESUMO
The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d
Assuntos
Quadruplex G , Pareamento de Bases , Dimerização , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Motivos de Nucleotídeos , Oligonucleotídeos/química , PrótonsRESUMO
Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.
Assuntos
Centrômero/química , DNA/química , Dípteros/genética , Heterocromatina/química , Larva/genética , Cromossomos Politênicos/química , Sequências de Repetição em Tandem/genética , Cromossomo X/química , Animais , Centrômero/genética , Mapeamento Cromossômico , DNA/genética , Heterocromatina/genética , Hibridização in Situ Fluorescente , Masculino , Meiose/genética , Dados de Sequência Molecular , Filogenia , Cromossomos Politênicos/genética , Glândulas Salivares/química , Glândulas Salivares/citologia , Fixação de Tecidos , Cromossomo X/genéticaRESUMO
The non-recombining Y chromosome is expected to degenerate over evolutionary time, however, gene gain is a common feature of Y chromosomes of mammals and Drosophila. Here, we report that a large palindrome containing interchromosomal segmental duplications is located in the vicinity of the first amplicon detected in the Y chromosome of D. melanogaster. The recent appearance of such amplicons suggests that duplications to the Y chromosome, followed by the amplification of the segmental duplications, are a mechanism for the continuing evolution of Drosophila Y chromosomes.
Assuntos
Drosophila melanogaster/genética , Duplicação Gênica , Genes de Insetos , Sequências Repetidas Invertidas , Cromossomo Y , Animais , Evolução Molecular , Modelos Genéticos , Dados de Sequência MolecularRESUMO
In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed "RaTART" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.
Assuntos
Cromossomos/química , Dípteros/genética , Retroelementos , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Cromossomos/ultraestrutura , DNA/química , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Glândulas Salivares/química , Telômero/ultraestruturaRESUMO
The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.
Assuntos
Centrômero/química , Drosophila melanogaster/genética , Evolução Molecular , Análise de Sequência de DNA/métodos , Telômero/química , Cromossomo Y/química , Animais , Cromossomos Artificiais Bacterianos , Clonagem Molecular , DNA/química , Elementos de DNA Transponíveis , Sequências Repetitivas de Ácido NucleicoRESUMO
The maintenance of terminal sequences is an important role of the telomere, since it prevents the loss of internal regions that encode essential genes. In most eukaryotes, this is accomplished by the telomerase. However, telomere length can also be maintained by other mechanisms, such as homologous recombination and transposition of telomeric retrotransposons to the chromosome ends. A remarkable situation is the case of Drosophila, where telomerase was lost, and thus telomeres managed to be maintained by occasional retrotransposition of telomeric elements to the receding ends. In the recent analysis of 12 Drosophila genomes, the multiplicity of autonomous and non-autonomous telomere-specific retrotransposons has revealed extensive and rapid evolution of telomeric DNA. The phylogenetic relationship among these telomeric retrotransposons is congruent with the species phylogeny, suggesting that they have been vertically transmitted from a common ancestor. In this review, we also suggest that the formation of a non-canonical DNA structure at Drosophila telomeres could be the way to protect the ends.
Assuntos
Drosophila/metabolismo , Evolução Molecular , Retroelementos/genética , Telômero/genética , Telômero/metabolismo , AnimaisRESUMO
The centromere is the region of the eukaryotic chromosome that determines kinetochore formation and sister chromatid cohesion. Centromeres interact with spindle microtubules to ensure chromatid segregation during mitosis and homologous chromosome segregation during meiosis I. In recent years, the overall organization of centromeres in several eukaryotic species has been described, yet the mechanisms of centromere definition remain elusive. Understanding the evolutionary origin of the centromere may well elucidate aspects of its function. With such intention, we hypothesize that centromeres were derived from telomeres during the evolution of the eukaryotic chromosome. We propose that the proto-eukaryotic cell could not have evolved a nucleus without concurrently evolving a new tubulin-based cytoskeleton, the microtubules and a specific chromosomal region that enabled the chromosome-microtubule interaction, the centromere. The repetitive nature of the subtelomeric regions that gave rise to the centromeres forced the concerted evolution of the centromeres. Although this implies the absence of a conserved primary sequence, a conserved centromere-specific structural motif could still exist and determine where in the chromosome the centromere is to be formed. To support the "centromeres-from-telomeres" hypothesis, we discuss several situations, in meiosis and mitosis, where telomeric regions took over centromeric roles. The recently discovered phenomenon of centromere repositioning is also discussed because it has revealed new insights into how neocentromeres evolve.
Assuntos
Evolução Biológica , Centrômero/genética , Animais , Divisão do Núcleo Celular , Centrômero/ultraestrutura , Células Eucarióticas/ultraestrutura , Telômero/genéticaRESUMO
Drosophila telomeres do not have arrays of simple telomerase-generated G-rich repeats. Instead, Drosophila maintains its telomeres by occasional transposition of specific non-long terminal repeat (non-LTR) retrotransposons to chromosome ends. The genus Drosophila provides a superb model system for comparative telomere analysis. Here we present an evolutionary study of Drosophila telomeric elements to ascertain the significance of telomeric retrotransposons (TRs) in the maintenance of Drosophila telomeres. PCR and in silico surveys in the sibling species of Drosophila melanogaster and in more distantly related species show that multiple TRs maintain telomeres in Drosophila. In addition to TRs with two open reading frames (ORFs) capable of autonomous transposition, there are deleted telomeric retrotransposons that have lost their ORF2, which we refer to as half telomeric-retrotransposons (HTRs). The phylogenetic relationship among these telomeric elements is congruent with the phylogeny of the species, suggesting that they have been vertically inherited from a common ancestor. Our results suggest that an existing non-LTR retrotransposon was recruited to perform the cellular function of telomere maintenance.
Assuntos
Drosophila/genética , Evolução Molecular , Retroelementos/genética , Telomerase/genética , Telômero/genética , Animais , Drosophila/enzimologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Genoma , Dados de Sequência Molecular , Filogenia , Especificidade da Espécie , Telômero/enzimologiaRESUMO
The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Metiltransferases/genética , Animais , Sequência de Bases , Centrômero/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Imuno-Histoquímica , Metiltransferases/metabolismo , Mutação/genética , Glândulas Salivares/metabolismoRESUMO
Genome sequences for most metazoans and plants are incomplete because of the presence of repeated DNA in the heterochromatin. The heterochromatic regions of Drosophila melanogaster contain 20 million bases (Mb) of sequence amenable to mapping, sequence assembly, and finishing. We describe the generation of 15 Mb of finished or improved heterochromatic sequence with the use of available clone resources and assembly methods. We also constructed a bacterial artificial chromosome-based physical map that spans 13 Mb of the pericentromeric heterochromatin and a cytogenetic map that positions 11 Mb in specific chromosomal locations. We have approached a complete assembly and mapping of the nonsatellite component of Drosophila heterochromatin. The strategy we describe is also applicable to generating substantially more information about heterochromatin in other species, including humans.
Assuntos
Drosophila melanogaster/genética , Heterocromatina/genética , Análise de Sequência de DNA , Animais , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Mapeamento de Sequências Contíguas , Genoma , Hibridização in Situ Fluorescente , Mapeamento Físico do CromossomoRESUMO
The centromere is the DNA region of the eukaryotic chromosome that determines kinetochore formation and sister chromatid cohesion. Centromeres interact with spindle microtubules to ensure the segregation of chromatids during mitosis and of homologous chromosomes in meiosis. The origin of centromeres, therefore, is inseparable from the evolution of cytoskeletal components that distribute chromosomes to offspring cells. Although the origin of the nucleus has been debated, no explanation for the evolutionary appearance of centromeres is available. We propose an evolutionary scenario: The centromeres originated from telomeres. The breakage of the ancestral circular genophore activated the transposition of retroelements at DNA ends that allowed the formation of telomeres by a recombination-dependent replication mechanism. Afterward, the modification of the tubulin-based cytoskeleton that allowed specific subtelomeric repeats to be recognized as new cargo gave rise to the first centromere. This switch from actin-based genophore partition to a tubulin-based mechanism generated a transition period during which both types of cytoskeleton contributed to fidelity of chromosome segregation. During the transition, pseudodicentric chromosomes increased the tendency toward chromosomal breakage and instability. This instability generated multiple telocentric chromosomes that eventually evolved into metacentric or holocentric chromosomes.
Assuntos
Evolução Biológica , Centrômero/genética , Cromossomos/genética , Células Eucarióticas/citologia , Telômero/genética , Modelos GenéticosRESUMO
Here we show that RNA interference (RNAi) machinery operates in Drosophila melanogaster 1.688 satellite transcription. Mutation in the spn-E gene, known to be involved in RNAi in the oocytes, causes an increase of satellite transcript abundance. Transcripts of both strands of 1.688 satellite repeats in germinal tissues were detected. The strength of the effects of the spn-E mutation differs for 1.688 satellite DNA subfamilies and is more pronounced for autosomal pericentromeric satellites compared to the X-linked centromeric ones. The spn-E(1) mutation causes an increase of the H3-AcK9 mark and TAF1 (a component of the polymerase II transcriptional complex) occupancy in the chromatin of autosomal pericentromeric repeats. Thus, we revealed that RNAi operates in ovaries to maintain the silenced state of centromeric and pericentromeric 1.688 repeats.