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1.
Cell ; 178(4): 964-979.e20, 2019 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-31398345

RESUMEN

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.


Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Argonautas/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , ARN Helicasas DEAD-box/metabolismo , Elementos Transponibles de ADN , Silenciador del Gen , Células Germinativas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética , Proteína Exportina 1
2.
Nature ; 628(8006): 122-129, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38448590

RESUMEN

Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7-9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.


Asunto(s)
Caenorhabditis , Impresión Genómica , ARN de Interacción con Piwi , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Femenino , Masculino , Alelos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis/genética , Caenorhabditis/metabolismo , Cruzamientos Genéticos , Padre , Genoma/genética , Impresión Genómica/genética , Organismos Hermafroditas/genética , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , Madres , Oocitos/metabolismo , ARN de Interacción con Piwi/genética , Biosíntesis de Proteínas , Secuencias Repetitivas de Ácidos Nucleicos/genética , ARN Mensajero/genética , Toxinas Biológicas/genética , Transcripción Genética
3.
Cell ; 157(6): 1364-1379, 2014 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-24906153

RESUMEN

Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 23-30 nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. Here, we show that a complex composed of Rhino, Deadlock, and Cutoff (RDC) defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC is anchored to H3K9me3-marked chromatin in part via Rhino's chromodomain. Depletion of Piwi results in loss of the RDC and small RNAs at a subset of piRNA clusters, demonstrating a feedback loop between Piwi and piRNA source loci. Intriguingly, profiles of RNA polymerase II occupancy, nascent transcription, and steady-state RNA levels reveal that the RDC licenses noncanonical transcription of dual-strand piRNA clusters. Likely, this process involves 5' end protection of nascent RNAs and suppression of transcription termination. Our data provide key insight into the regulation and evolution of piRNA clusters.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Animales , Femenino , Estudio de Asociación del Genoma Completo , Ovario/metabolismo , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Terminación de la Transcripción Genética
4.
Genes Dev ; 35(5-6): 392-409, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33574069

RESUMEN

Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.


Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Silenciador del Gen/fisiología , Complejos Multiproteicos/metabolismo , Animales , Dimerización , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
5.
Nat Methods ; 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-38969721

RESUMEN

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

6.
Cell ; 151(5): 964-80, 2012 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-23159368

RESUMEN

Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Silenciador del Gen , ARN Interferente Pequeño/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Elementos Transponibles de ADN , Heterocromatina/metabolismo , Código de Histonas
7.
Cell ; 137(3): 522-35, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19395010

RESUMEN

In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.


Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ovario/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Animales , Femenino , Silenciador del Gen , Mutación , Ovario/citología , ARN no Traducido/genética , ARN no Traducido/metabolismo , Retroelementos
8.
Nature ; 549(7670): 54-59, 2017 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-28847004

RESUMEN

Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. Here we show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters-small RNA source loci in animal gonads-is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.


Asunto(s)
Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Heterocromatina/metabolismo , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/genética , Transcripción Genética , Animales , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Silenciador del Gen , Heterocromatina/química , Familia de Multigenes/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/química , ARN Interferente Pequeño/biosíntesis , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Factor de Transcripción TFIIA/metabolismo , Iniciación de la Transcripción Genética
9.
Proc Natl Acad Sci U S A ; 117(48): 30370-30379, 2020 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-33199607

RESUMEN

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/ß-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.


Asunto(s)
Región de Flanqueo 3' , Proteínas de Drosophila/química , Exorribonucleasas/química , MicroARNs/química , MicroARNs/genética , Procesamiento Postranscripcional del ARN , Relación Estructura-Actividad , Animales , Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Exorribonucleasas/metabolismo , MicroARNs/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo
10.
Genes Dev ; 29(16): 1747-62, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-26302790

RESUMEN

PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts­including those from piRNA clusters­as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.


Asunto(s)
Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Silenciador del Gen , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Ovario/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Postranscripcional del ARN
11.
Genes Dev ; 29(21): 2258-71, 2015 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-26494711

RESUMEN

The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.


Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , ADN/metabolismo , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Femenino , Silenciador del Gen , Genoma de los Insectos/genética , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Metilación , Ovario/fisiología , Unión Proteica , ARN/metabolismo , Proteínas de Unión al ARN , Proteínas Represoras/metabolismo
12.
Nature ; 539(7630): 588-592, 2016 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-27851737

RESUMEN

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)-the 22-30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads-are generated independently of Dicer from single-stranded precursors. piRNA 5' ends are defined either by Zucchini, the Drosophila homologue of mitoPLD-a mitochondria-anchored endonuclease, or by piRNA-guided target cleavage. Formation of piRNA 3' ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3'-end formation.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Animales , Proteínas Argonautas/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/enzimología , Drosophila melanogaster/metabolismo , Endorribonucleasas/deficiencia , Endorribonucleasas/metabolismo , Exorribonucleasas/deficiencia , Exorribonucleasas/metabolismo , Femenino , Proteínas Nucleares/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Postranscripcional del ARN , ARN Guía de Kinetoplastida/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Transcripción Genética
13.
Nucleic Acids Res ; 48(14): e79, 2020 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-32496553

RESUMEN

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.


Asunto(s)
Proteínas Argonautas/metabolismo , Cromatografía Liquida/métodos , ARN Interferente Pequeño/aislamiento & purificación , Complejo Silenciador Inducido por ARN/química , Animales , Resinas de Intercambio Aniónico , Proteínas Argonautas/aislamiento & purificación , Línea Celular Tumoral , Biblioteca de Genes , Ratones , Ratones Endogámicos C57BL , Polinucleótido 5'-Hidroxil-Quinasa , ARN de Hongos/aislamiento & purificación , ARN de Helminto/aislamiento & purificación , ARN Neoplásico/aislamiento & purificación , ARN de Planta/aislamiento & purificación , ARN Protozoario/aislamiento & purificación , ARN Interferente Pequeño/sangre , ARN Interferente Pequeño/metabolismo , Sefarosa , Dióxido de Silicio , Ultracentrifugación
14.
Genes Dev ; 28(16): 1772-85, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-25081352

RESUMEN

Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Intrones/genética , Empalme del ARN/fisiología , Animales , Proteínas Argonautas/genética , Células Cultivadas , Elementos Transponibles de ADN/genética , Drosophila/clasificación , Proteínas de Drosophila/genética , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Ovario/metabolismo , Precursores del ARN/metabolismo
15.
Mol Cell ; 50(5): 762-77, 2013 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-23665231

RESUMEN

The piRNA (PIWI-interacting RNA) pathway is a small RNA silencing system that acts in animal gonads and protects the genome against the deleterious influence of transposons. A major bottleneck in the field is the lack of comprehensive knowledge of the factors and molecular processes that constitute this pathway. We conducted an RNAi screen in Drosophila and identified ~50 genes that strongly impact the ovarian somatic piRNA pathway. Many identified genes fall into functional categories that indicate essential roles for mitochondrial metabolism, RNA export, the nuclear pore, transcription elongation, and chromatin regulation in the pathway. Follow-up studies on two factors demonstrate that components acting at distinct hierarchical levels of the pathway were identified. Finally, we define CG2183/Gasz as an essential primary piRNA biogenesis factor in somatic and germline cells. Based on the similarities between insect and vertebrate piRNA pathways, our results have far-reaching implications for the understanding of this conserved genome defense system.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Ovario/fisiología , ARN Interferente Pequeño/genética , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Femenino , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Sensibilidad y Especificidad
16.
Genes Dev ; 27(15): 1693-705, 2013 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-23913922

RESUMEN

The PIWI-interacting RNA (piRNA) pathway is a small RNA silencing system that keeps selfish genetic elements such as transposons under control in animal gonads. Several lines of evidence indicate that nuclear PIWI family proteins guide transcriptional silencing of their targets, yet the composition of the underlying silencing complex is unknown. Here we demonstrate that the double CHHC zinc finger protein gametocyte-specific factor 1 (Gtsf1) is an essential factor for Piwi-mediated transcriptional repression in Drosophila. Cells lacking Gtsf1 contain nuclear Piwi loaded with piRNAs, yet Piwi's silencing capacity is ablated. Gtsf1 interacts directly with a small subpool of nuclear Piwi, and loss of Gtsf1 phenocopies loss of Piwi in terms of deregulation of transposons, loss of H3K9 trimethylation (H3K9me3) marks at euchromatic transposon insertions, and deregulation of genes in proximity to repressed transposons. We propose that only a small fraction of nuclear Piwi is actively engaged in target silencing and that Gtsf1 is an essential component of the underlying Piwi-centered silencing complex.


Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Complejo Mediador/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Células Cultivadas , Femenino , Silenciador del Gen , Ovario/metabolismo
17.
Mol Cell ; 47(6): 954-69, 2012 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-22902557

RESUMEN

In animal gonads, PIWI proteins and their bound 23-30 nt piRNAs guard genome integrity by the sequence specific silencing of transposons. Two branches of piRNA biogenesis, namely primary processing and ping-pong amplification, have been proposed. Despite an overall conceptual understanding of piRNA biogenesis, identity and/or function of the involved players are largely unknown. Here, we demonstrate an essential role for the female sterility gene shutdown in piRNA biology. Shutdown, an evolutionarily conserved cochaperone collaborates with Hsp90 during piRNA biogenesis, potentially at the loading step of RNAs into PIWI proteins. We demonstrate that Shutdown is essential for both primary and secondary piRNA populations in Drosophila. An extension of our study to previously described piRNA pathway members revealed three distinct groups of biogenesis factors. Together with data on how PIWI proteins are wired into primary and secondary processing, we propose a unified model for piRNA biogenesis.


Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , ARN Interferente Pequeño/genética , Animales , Células Cultivadas , Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo
18.
Mol Cell ; 36(3): 445-56, 2009 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-19917252

RESUMEN

Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. MicroRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a byproduct of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de Drosophila/metabolismo , MicroARNs/metabolismo , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Regiones no Traducidas 3' , Animales , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Emparejamiento Base , Northern Blotting , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inmunoprecipitación , MicroARNs/química , MicroARNs/genética , Modelos Biológicos , Conformación de Ácido Nucleico , Unión Proteica , Interferencia de ARN , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Complejo Silenciador Inducido por ARN/genética , Termodinámica
19.
EMBO J ; 30(19): 3977-93, 2011 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-21863019

RESUMEN

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb.


Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Alelos , Animales , Cruzamientos Genéticos , Elementos Transponibles de ADN , Drosophila melanogaster , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ovario/metabolismo , Estructura Terciaria de Proteína , ARN/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética
20.
EMBO J ; 29(19): 3301-17, 2010 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-20818334

RESUMEN

In Drosophila, PIWI proteins and bound PIWI-interacting RNAs (piRNAs) form the core of a small RNA-mediated defense system against selfish genetic elements. Within germline cells, piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target-dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi-mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi-nuclear accumulation. We show that Armitage and Yb interact physically and co-localize in cytoplasmic Yb bodies, which flank P bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb bodies, indicating that Yb bodies are sites of primary piRNA biogenesis.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Endorribonucleasas/metabolismo , Folículo Ovárico/metabolismo , ARN Helicasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/biosíntesis , Transporte Activo de Núcleo Celular/fisiología , Animales , Células Cultivadas , Cartilla de ADN/genética , Elementos Transponibles de ADN/genética , Femenino , Inmunohistoquímica , Inmunoprecipitación , Luciferasas , Reacción en Cadena de la Polimerasa
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