RESUMO
From nematodes to placental mammals, key components of the germline transposon silencing piRNAs pathway localize to phase separated perinuclear granules. In Drosophila, the PIWI protein Aub, DEAD box protein Vasa and helicase Armi localize to nuage granules and are required for ping-pong piRNA amplification and phased piRNA processing. Drosophila piRNA mutants lead to genome instability and Chk2 kinase DNA damage signaling. By systematically analyzing piRNA pathway organization, small RNA production, and long RNA expression in single piRNA mutants and corresponding chk2/mnk double mutants, we show that Chk2 activation disrupts nuage localization of Aub and Vasa, and that the HP1 homolog Rhino, which drives piRNA precursor transcription, is required for Aub, Vasa, and Armi localization to nuage. However, these studies also show that ping-pong amplification and phased piRNA biogenesis are independent of nuage localization of Vasa, Aub and Armi. Dispersed cytoplasmic proteins thus appear to mediate these essential piRNA pathway functions.
RESUMO
Transposons are mobile genetic elements prevalent in the genomes of most species. The distribution of transposons within a genome reflects the actions of two opposing processes: initial insertion site selection, and selective pressure from the host. By analyzing whole-genome sequencing data from transposon-activated Drosophila melanogaster, we identified 43 316 de novo and 237 germline insertions from four long-terminal-repeat (LTR) transposons, one LINE transposon (I-element), and one DNA transposon (P-element). We found that all transposon types favored insertion into promoters de novo, but otherwise displayed distinct insertion patterns. De novo and germline P-element insertions preferred replication origins, often landing in a narrow region around transcription start sites and in regions of high chromatin accessibility. De novo LTR transposon insertions preferred regions with high H3K36me3, promoters and exons of active genes; within genes, LTR insertion frequency correlated with gene expression. De novo I-element insertion density increased with distance from the centromere. Germline I-element and LTR transposon insertions were depleted in promoters and exons, suggesting strong selective pressure to remove transposons from functional elements. Transposon movement is associated with genome evolution and disease; therefore, our results can improve our understanding of genome and disease biology.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos de DNA Transponíveis/genética , Cromossomos , Sequência de Bases , Epigênese GenéticaRESUMO
piRNAs guide Piwi/Panoramix-dependent H3K9me3 chromatin modification and transposon silencing during Drosophila germline development. The THO RNA export complex is composed of Hpr1, Tho2, and Thoc5-7. Null thoc7 mutations, which displace Thoc5 and Thoc6 from a Tho2-Hpr1 subcomplex, reduce expression of a subset of germline piRNAs and increase transposon expression, suggesting that THO silences transposons by promoting piRNA biogenesis. Here, we show that the thoc7-null mutant combination increases transposon transcription but does not reduce anti-sense piRNAs targeting half of the transcriptionally activated transposon families. These mutations also fail to reduce piRNA-guided H3K9me3 chromatin modification or block Panoramix-dependent silencing of a reporter transgene, and unspliced transposon transcripts co-precipitate with THO through a Piwi- and Panoramix-independent mechanism. Mutations in piwi also dominantly enhance germline defects associated with thoc7-null alleles. THO thus functions in a piRNA-independent transposon-silencing pathway, which acts cooperatively with Piwi to support germline development.
Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica/fisiologia , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , Animais , Proteínas Argonautas/genética , Núcleo Celular/metabolismo , Elementos de DNA Transponíveis/genética , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismoRESUMO
Transposons are genomic parasites, and their new insertions can cause instability and spur the evolution of their host genomes. Rapid accumulation of short-read whole-genome sequencing data provides a great opportunity for studying new transposon insertions and their impacts on the host genome. Although many algorithms are available for detecting transposon insertions, the task remains challenging and existing tools are not designed for identifying de novo insertions. Here, we present a new benchmark fly dataset based on PacBio long-read sequencing and a new method TEMP2 for detecting germline insertions and measuring de novo 'singleton' insertion frequencies in eukaryotic genomes. TEMP2 achieves high sensitivity and precision for detecting germline insertions when compared with existing tools using both simulated data in fly and experimental data in fly and human. Furthermore, TEMP2 can accurately assess the frequencies of de novo transposon insertions even with high levels of chimeric reads in simulated datasets; such chimeric reads often occur during the construction of short-read sequencing libraries. By applying TEMP2 to published data on hybrid dysgenic flies inflicted by de-repressed P-elements, we confirmed the continuous new insertions of P-elements in dysgenic offspring before they regain piRNAs for P-element repression. TEMP2 is freely available at Github: https://github.com/weng-lab/TEMP2.
Assuntos
Benchmarking/métodos , Elementos de DNA Transponíveis , Drosophila/genética , Genômica/métodos , Células Germinativas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Animais , Feminino , Genoma Humano , Humanos , Software , Sequenciamento Completo do GenomaRESUMO
In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.
Assuntos
Epigênese Genética , Éxons/genética , Mamíferos/genética , Estágio Paquíteno/genética , RNA Interferente Pequeno/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilação , Animais , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Histonas/metabolismo , Íntrons/genética , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Testículo/metabolismo , Transcrição GênicaRESUMO
PIWI-interacting RNAs (piRNAs) are small single-stranded RNAs that can repress transposon expression via epigenetic silencing and transcript degradation. They have been identified predominantly in the ovary and testis, where they serve essential roles in transposon silencing in order to protect the integrity of the genome in the germline. The potential expression of piRNAs in somatic cells has been controversial. In the present study we demonstrate the expression of piRNAs derived from both genic and transposon RNAs in the intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta. These piRNAs are abundantly expressed, â¼27 nt long, map antisense to transposons, are oxidation resistant, exhibit a 5' uridine bias, and amplify via the canonical ping-pong pathway. An RNA-seq analysis demonstrated that 19 piRNA pathway genes are expressed in the ISMs and are developmentally regulated. The abundance of piRNAs does not change when the muscles initiate developmentally-regulated atrophy, but are repressed coincident with the commitment of the muscles undergo programmed cell death at the end of metamorphosis. This change in piRNA expression is correlated with the repression of several retrotransposons and the induction of specific DNA transposons. The developmentally-regulated changes in the expression of piRNAs, piRNA pathway genes, and transposons are all regulated by 20-hydroxyecdysone, the steroid hormone that controls the timing of ISM death. Taken together, these data provide compelling evidence for the existence of piRNA in somatic tissues and suggest that they may play roles in developmental processes such as programmed cell death.
RESUMO
Skeletal muscles can undergo atrophy and/or programmed cell death (PCD) during development or in response to a wide range of insults, including immobility, cachexia, and spinal cord injury. However, the protracted nature of atrophy and the presence of multiple cell types within the tissue complicate molecular analyses. One model that does not suffer from these limitations is the intersegmental muscle (ISM) of the tobacco hawkmoth Manduca sexta. Three days before the adult eclosion (emergence) at the end of metamorphosis, the ISMs initiate a nonpathological program of atrophy that results in a 40% loss of mass. The ISMs then generate the eclosion behavior and initiate a nonapoptotic PCD during the next 30 h. We have performed a comprehensive transcriptomics analysis of all mRNAs and microRNAs throughout ISM development to better understand the molecular mechanisms that mediate atrophy and death. Atrophy involves enhanced protein catabolism and reduced expression of the genes involved in respiration, adhesion, and the contractile apparatus. In contrast, PCD involves the induction of numerous proteases, DNA methylases, membrane transporters, ribosomes, and anaerobic metabolism. These changes in gene expression are largely repressed when insects are injected with the insect steroid hormone 20-hydroxyecdysone, which delays death. The expression of the death-associated proteins may be greatly enhanced by reductions in specific microRNAs that function to repress translation. This study not only provides fundamental new insights into basic developmental processes, it may also represent a powerful resource for identifying potential diagnostic markers and molecular targets for therapeutic intervention.
Assuntos
Apoptose/genética , Genes de Insetos , Manduca/genética , Atrofia Muscular/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Contráteis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , MicroRNAs/genética , Contração Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , RNA Mensageiro/genéticaRESUMO
In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription.
Assuntos
Drosophila/genética , Redes Reguladoras de Genes , RNA Interferente Pequeno/metabolismo , Alelos , Animais , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Genes Dominantes , Modelos Biológicos , Mutação/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.
Assuntos
Gammaretrovirus/genética , Phascolarctidae/genética , RNA Interferente Pequeno/genética , Animais , Elementos de DNA Transponíveis , Gammaretrovirus/metabolismo , Gammaretrovirus/patogenicidade , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Genoma , Células Germinativas/metabolismo , Células Germinativas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Phascolarctidae/virologia , Splicing de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Transposons are major genome constituents that can mobilize and trigger mutations, DNA breaks and chromosome rearrangements. Transposon silencing is particularly important in the germline, which is dedicated to transmission of the inherited genome. Piwi-interacting RNAs (piRNAs) guide a host defence system that transcriptionally and post-transcriptionally silences transposons during germline development. While germline control of transposons by the piRNA pathway is conserved, many piRNA pathway genes are evolving rapidly under positive selection, and the piRNA biogenesis machinery shows remarkable phylogenetic diversity. Conservation of core function combined with rapid gene evolution is characteristic of a host-pathogen arms race, suggesting that transposons and the piRNA pathway are engaged in an evolutionary tug of war that is driving divergence of the biogenesis machinery. Recent studies suggest that this process may produce biochemical incompatibilities that contribute to reproductive isolation and species divergence.
Assuntos
Evolução Molecular , Inativação Gênica , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Animais , Elementos de DNA Transponíveis/genética , Variação Genética , Humanos , Modelos Genéticos , Seleção GenéticaRESUMO
In Drosophila, the piRNAs that guide germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. We show that Rhino promotes cluster transcript association with UAP56 and the THO complex, forming RNA-protein assemblies that are unique to piRNA precursors. UAP56 and THO are ubiquitous RNA-processing factors, and null alleles of uap56 and the THO subunit gene tho2 are lethal. However, uap56sz15 and mutations in the THO subunit genes thoc5 and thoc7 are viable but sterile and disrupt piRNA biogenesis. The uap56sz15 allele reduces UAP56 binding to THO, and the thoc5 and thoc7 mutations disrupt interactions among the remaining THO subunits and UAP56 binding to the core THO subunit Hpr1. These mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino at cluster heterochromatin.
Assuntos
Heterocromatina/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Sítios de Ligação , Proteínas Cromossômicas não Histona/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Heterocromatina/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
PIWI-interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species-specific manner and so defines the piRNA-producing loci in the Drosophila genome. Here, we determine the crystal structures of Rhino-Deadlock complex in Drosophila melanogaster and simulans In both species, one Rhino binds the N-terminal helix-hairpin-helix motif of one Deadlock protein through a novel interface formed by the beta-sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. Our structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross-species incompatibility between the sibling species. By determining the molecular architecture of this piRNA-producing machinery, we discover a novel HP1-partner interacting mode that is crucial to piRNA biogenesis and transposon silencing. We thus explain the cross-species incompatibility of two sibling species at the molecular level.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Drosophila/química , Proteínas Associadas aos Microtúbulos/química , Complexos Multiproteicos/química , RNA Interferente Pequeno/genética , Animais , Proteínas Cromossômicas não Histona/genética , Cristalografia por Raios X , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma de Inseto/genética , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos/genética , RNA Interferente Pequeno/química , Especificidade da EspécieRESUMO
The ability to integrate experiential information and recall it in the form of memory is observed in a wide range of taxa, and is a hallmark of highly derived nervous systems. Storage of past experiences is critical for adaptive behaviors that anticipate both adverse and positive environmental factors. The process of memory formation and consolidation involve many synchronized biological events including gene transcription, protein modification, and intracellular trafficking: However, many of these molecular mechanisms remain illusive. With Drosophila as a model system we use a nonassociative memory paradigm and a systems level approach to uncover novel transcriptional patterns. RNA sequencing of Drosophila heads during and after memory formation identified a number of novel memory genes. Tracking the dynamic expression of these genes over time revealed complex gene networks involved in long term memory. In particular, this study focuses on two functional gene clusters of signal peptides and proteases. Bioinformatics network analysis and prediction in combination with high-throughput RNA sequencing identified previously unknown memory genes, which when genetically knocked down resulted in behaviorally validated memory defects.
Assuntos
Drosophila/genética , Redes Reguladoras de Genes/genética , Memória de Longo Prazo/fisiologia , Animais , Biologia Computacional/métodos , Proteínas de Drosophila/genética , Modelos Animais , Análise de Sequência de RNA/métodosRESUMO
Reproductive isolation defines species divergence and is linked to adaptive evolution of hybrid incompatibility genes. Hybrids between Drosophila melanogaster and Drosophila simulans are sterile, and phenocopy mutations in the PIWI interacting RNA (piRNA) pathway, which silences transposons and shows pervasive adaptive evolution, and Drosophila rhino and deadlock encode rapidly evolving components of a complex that binds to piRNA clusters. We show that Rhino and Deadlock interact and co-localize in simulans and melanogaster, but simulans Rhino does not bind melanogaster Deadlock, due to substitutions in the rapidly evolving Shadow domain. Significantly, a chimera expressing the simulans Shadow domain in a melanogaster Rhino backbone fails to support piRNA production, disrupts binding to piRNA clusters, and leads to ectopic localization to bulk heterochromatin. Fusing melanogaster Deadlock to simulans Rhino, by contrast, restores localization to clusters. Deadlock binding thus directs Rhino to piRNA clusters, and Rhino-Deadlock co-evolution has produced cross-species incompatibilities, which may contribute to reproductive isolation.
Assuntos
Elementos de DNA Transponíveis/genética , Drosophila melanogaster/metabolismo , Inativação Gênica/fisiologia , RNA Interferente Pequeno/genética , Animais , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Heterocromatina/metabolismo , Fenótipo , Isolamento ReprodutivoRESUMO
piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and speculate that stalled splicing differentiates piRNA precursors from mRNAs.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Splicing de RNA , RNA Interferente Pequeno/genética , Animais , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Ovário/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXD/genéticaRESUMO
Uncontrolled transposable element (TE) insertions and excisions can cause chromosome breaks and mutations with dramatic deleterious effects. The PIWI interacting RNA (piRNA) pathway functions as an adaptive TE silencing system during germline development. Several essential piRNA pathway proteins appear to be rapidly evolving, suggesting that TEs and the silencing machinery may be engaged in a classical "evolutionary arms race." Using a variety of molecular evolutionary and population genetic approaches, we find that the piRNA pathway genes rhino, krimper, and aubergine show patterns suggestive of extensive recurrent positive selection across Drosophila species. We speculate that selection on these proteins reflects crucial roles in silencing unfamiliar elements during vertical and horizontal transmission of TEs into naïve populations and species, respectively.
Assuntos
Drosophila/genética , Evolução Molecular , RNA Interferente Pequeno/genética , Seleção Genética , Animais , Drosophila/classificação , Genes de Insetos/genética , Células Germinativas/metabolismo , Filogenia , População/genéticaRESUMO
piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Dano ao DNA , Elementos de DNA Transponíveis , Feminino , Células Germinativas/citologia , Masculino , Membrana Nuclear/metabolismoRESUMO
BACKGROUND: High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs. DESIGN: Here, we describe a procedure for preparing RNA-Seq libraries from 1 to 4 µg total RNA without poly(A) selection. Our method combines the deoxyuridine triphosphate (dUTP)/uracil-DNA glycosylase (UDG) strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. We barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms. DISCUSSION: The RNA-Seq protocol described here yields strand-specific transcriptome libraries without poly(A) selection, which provide approximately 90% mappable sequences. Typically, more than 85% of mapped reads correspond to protein-coding genes and only 6% derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability.
RESUMO
Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.