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1.
Nat Struct Mol Biol ; 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38658622

RESUMO

The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host-pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate-which enforces seed-target fidelity in microRNA RISC-adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed-target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA-target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species.

3.
Cell Rep ; 40(10): 111265, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36070689

RESUMO

Germline Argonautes direct transcriptome surveillance within perinuclear membraneless organelles called nuage. In C. elegans, a family of Vasa-related Germ Line Helicase (GLH) proteins localize in and promote the formation of nuage. Previous studies have implicated GLH proteins in inherited silencing, but direct roles in small-RNA production, Argonaute binding, or mRNA targeting have not been identified. Here we show that GLH proteins compete with each other to control Argonaute pathway specificity, bind directly to Argonaute target mRNAs, and promote the amplification of small RNAs required for transgenerational inheritance. We show that the ATPase cycle of GLH-1 regulates direct binding to the Argonaute WAGO-1, which engages amplified small RNAs. Our findings support a dynamic and direct role for GLH proteins in inherited silencing beyond their role as structural components of nuage.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , RNA Helicases DEAD-box/metabolismo , Células Germinativas/metabolismo , RNA Mensageiro/metabolismo
4.
Cell Res ; 32(11): 969-981, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36104507

RESUMO

The dynamic three-dimensional structures of chromatin and extrachromosomal DNA molecules regulate fundamental cellular processes and beyond. However, the visualization of specific DNA sequences in live cells, especially nonrepetitive sequences accounting for most of the genome, is still vastly challenging. Here, we introduce a robust CRISPR-mediated fluorescence in situ hybridization amplifier (CRISPR FISHer) system, which exploits engineered sgRNA and protein trimerization domain-mediated, phase separation-based exponential assembly of fluorescent proteins in the CRISPR-targeting locus, conferring enhancements in both local brightness and signal-to-background ratio and thus achieving single sgRNA-directed visualization of native nonrepetitive DNA loci in live cells. In one application, by labeling and tracking the broken ends of chromosomal fragments, CRISPR FISHer enables real-time visualization of the entire process of chromosome breakage, separation, and subsequent intra- or inter-chromosomal ends rejoining in a single live cell. Furthermore, CRISPR FISHer allows the movement of small extrachromosomal circular DNAs (eccDNAs) and invading DNAs to be recorded, revealing substantial differences in dynamic behaviors between chromosomal and extrachromosomal loci. With the potential to track any specified self or non-self DNA sequences, CRISPR FISHer dramatically broadens the scope of live-cell imaging in biological events and for biomedical diagnoses.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Hibridização in Situ Fluorescente , DNA/metabolismo , Cromatina , Genoma , Sistemas CRISPR-Cas/genética
6.
Mol Cell ; 70(4): 639-649.e6, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29775580

RESUMO

Animal cells have a remarkable capacity to adopt durable and heritable gene expression programs or epigenetic states that define the physical properties and diversity of somatic cell types. The maintenance of epigenetic programs depends on poorly understood pathways that prevent gain or loss of inherited signals. In the germline, epigenetic factors are enriched in liquid-like perinuclear condensates called nuage. Here, we identify the deeply conserved helicase-domain protein, ZNFX-1, as an epigenetic regulator and component of nuage that interacts with Argonaute systems to balance epigenetic inheritance. Our findings suggest that ZNFX-1 promotes the 3' recruitment of machinery that propagates the small RNA epigenetic signal and thus counteracts a tendency for Argonaute targeting to shift 5' along the mRNA. These functional insights support the idea that recently identified subdomains of nuage, including ZNFX-1 granules or "Z-granules," may define spatial and temporal zones of molecular activity during epigenetic regulation.


Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Núcleo Celular/genética , Epigênese Genética , Células Germinativas/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , Animais , Proteínas Argonautas/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Organelas , RNA Helicases/genética , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo
7.
Cell ; 172(5): 937-951.e18, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29456082

RESUMO

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , RNA Interferente Pequeno/metabolismo , Sequência de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteínas de Caenorhabditis elegans/química , Quimera/metabolismo , Inativação Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Cell Rep ; 22(9): 2254-2264, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29456100

RESUMO

Protein-coding genes undergo a wide array of regulatory interactions with factors that engage non-coding regions. Open reading frames (ORFs), in contrast, are thought to be constrained by coding function, precluding a major role in gene regulation. Here, we explore Piwi-interacting (pi)RNA-mediated transgene silencing in C. elegans and show that marked differences in the sensitivity to piRNA silencing map to the endogenous sequences within transgene ORFs. Artificially increasing piRNA targeting within the ORF of a resistant transgene can lead to a partial yet stable reduction in expression, revealing that piRNAs not only silence but can also "tune" gene expression. Our findings support a model that involves a temporal element to mRNA regulation by germline Argonautes, likely prior to translation, and suggest that piRNAs afford incremental control of germline mRNA expression by targeting the body of the mRNA, including the coding region.


Assuntos
Proteínas Argonautas/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Fases de Leitura Aberta/genética , Animais , Proteínas Argonautas/metabolismo , Sequência de Bases , Proteínas de Caenorhabditis elegans/metabolismo , Códon sem Sentido/genética , Inativação Gênica , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transgenes
9.
Dev Cell ; 44(6): 762-770.e3, 2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29456136

RESUMO

In metazoans, Piwi-related Argonaute proteins engage piRNAs (Piwi-interacting small RNAs) to defend the genome against invasive nucleic acids, such as transposable elements. Yet many organisms-including worms and humans-express thousands of piRNAs that do not target transposons, suggesting that piRNA function extends beyond genome defense. Here, we show that the X chromosome-derived piRNA 21ux-1 downregulates XOL-1 (XO Lethal), a master regulator of X chromosome dosage compensation and sex determination in Caenorhabditis elegans. Mutations in 21ux-1 and several Piwi-pathway components sensitize hermaphrodites to dosage compensation and sex determination defects. We show that the piRNA pathway also targets xol-1 in C. briggsae, a nematode species related to C. elegans. Our findings reveal physiologically important piRNA-mRNA interactions, raising the possibility that piRNAs function broadly to ensure robust gene expression and germline development.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Mecanismo Genético de Compensação de Dose , Regulação da Expressão Gênica , RNA Interferente Pequeno/genética , Cromossomos Sexuais , Análise para Determinação do Sexo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Fenótipo
10.
Nature ; 508(7494): 128-32, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24522532

RESUMO

It has been theorized for decades that mitochondria act as the biological clock of ageing, but the evidence is incomplete. Here we show a strong coupling between mitochondrial function and ageing by in vivo visualization of the mitochondrial flash (mitoflash), a frequency-coded optical readout reflecting free-radical production and energy metabolism at the single-mitochondrion level. Mitoflash activity in Caenorhabditis elegans pharyngeal muscles peaked on adult day 3 during active reproduction and on day 9 when animals started to die off. A plethora of genetic mutations and environmental factors inversely modified the lifespan and the day-3 mitoflash frequency. Even within an isogenic population, the day-3 mitoflash frequency was negatively correlated with the lifespan of individual animals. Furthermore, enhanced activity of the glyoxylate cycle contributed to the decreased day-3 mitoflash frequency and the longevity of daf-2 mutant animals. These results demonstrate that the day-3 mitoflash frequency is a powerful predictor of C. elegans lifespan across genetic, environmental and stochastic factors. They also support the notion that the rate of ageing, although adjustable in later life, has been set to a considerable degree before reproduction ceases.


Assuntos
Caenorhabditis elegans/metabolismo , Longevidade , Mitocôndrias/metabolismo , Superóxidos/metabolismo , Envelhecimento/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Morte , Metabolismo Energético , Meio Ambiente , Glioxilatos/metabolismo , Organismos Hermafroditas , Longevidade/genética , Longevidade/fisiologia , Masculino , Modelos Biológicos , Músculos/citologia , Mutação , Estresse Oxidativo , Receptor de Insulina/genética , Reprodução , Processos Estocásticos , Superóxidos/análise , Fatores de Tempo
11.
PLoS Genet ; 9(8): e1003715, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23950735

RESUMO

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12-Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.


Assuntos
Autofagia/genética , Proteínas de Membrana/genética , Peptídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas Relacionadas à Autofagia , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA , Genoma Fúngico , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Saccharomyces cerevisiae , Deleção de Sequência , Vacúolos
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