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/metabolismoRESUMO
During each life cycle, germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here, we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together, these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small RNA complexes that constitute a memory of gene expression in preceding generations.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Epigênese Genética , Proteínas de Ligação a RNA/metabolismo , Espermatogênese , Animais , Caenorhabditis elegans/genética , Feminino , Masculino , Pequeno RNA não Traduzido/metabolismo , Transdução de Sinais , Espermatozoides , Transcrição GênicaRESUMO
Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci; only one C. elegans transposon is a known piRNA target. Here, we show that, in mutants lacking the Piwi Argonaute PRG-1 (and consequently its associated piRNAs/21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression with a concomitant depletion of RNA-dependent RNA polymerase (RdRP)-derived secondary small RNAs termed 22G-RNAs. Sequences depleted of 22G-RNAs are proximal to potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Genoma Helmíntico , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/metabolismo , Inativação Gênica , Células GerminativasRESUMO
Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish "self" from "nonself" nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of "self" and "nonself" RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.
Assuntos
Caenorhabditis elegans/genética , Epigenômica , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inativação Gênica , Células Germinativas/metabolismo , Interferência de RNARESUMO
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/metabolismoRESUMO
RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Animais , Caenorhabditis elegans/genética , RNA Helicases DEAD-box/metabolismo , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Genoma/genética , Células Germinativas/metabolismo , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/química , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Análise de Sequência de RNA , TemperaturaRESUMO
In early Caenorhabditis elegans embryos, the Wingless/int (Wnt)- and Src-signaling pathways function in parallel to induce both the division orientation of the endomesoderm (EMS) blastomere and the endoderm fate of the posterior EMS daughter cell, called E. Here, we show that, in addition to its role in endoderm specification, the ß-catenin-related protein Worm armadillo 1 (WRM-1) also plays a role in controlling EMS division orientation. WRM-1 localizes to the cortex of cells in both embryos and larvae and is released from the cortex in a Wnt-responsive manner. We show that WRM-1 cortical release is disrupted in a hypomorphic cyclin-dependent protein kinase 1 (cdk-1) mutant and that WRM-1 lacking potential CDK-1 phosphoacceptor sites is retained at the cortex. In both cases, cortical WRM-1 interferes with EMS spindle rotation without affecting endoderm specification. Finally, we show that removal of WRM-1 from the cortex can restore WT division orientation, even when both Wnt- and Src-signaling pathways are compromised. Our findings are consistent with a model in which Wnt signaling and CDK-1 modify WRM-1 in a temporal and spatial manner to unmask an intrinsic polarity cue required for proper orientation of the EMS cell division axis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Divisão Celular/genética , Divisão Celular/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Genes de Helmintos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Prófase/genética , Prófase/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fuso Acromático/metabolismo , Via de Sinalização Wnt , Quinases da Família src/metabolismoRESUMO
Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 26G-RNAs which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Fertilidade/fisiologia , RNA Interferente Pequeno/biossíntese , Proteínas de Ligação a RNA/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Fertilidade/genética , Temperatura Alta , Masculino , Mutação , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Espermatogênese/genéticaRESUMO
Argonaute/small RNA pathways and heterochromatin work together to propagate transgenerational gene silencing, but the mechanisms behind their interaction are not well understood. Here, we show that induction of heterochromatin silencing in C. elegans by RNAi or by artificially tethering pathway components to target RNA causes co-localization of target alleles in pachytene nuclei. Tethering the nuclear Argonaute WAGO-9/HRDE-1 induces heterochromatin formation and independently induces small RNA amplification. Consistent with this finding, HRDE-1, while predominantly nuclear, also localizes to peri-nuclear nuage domains, where amplification is thought to occur. Tethering a heterochromatin-silencing factor, NRDE-2, induces heterochromatin formation, which subsequently causes de novo synthesis of HRDE-1 guide RNAs. HRDE-1 then acts to further amplify small RNAs that load on downstream Argonautes. These findings suggest that HRDE-1 plays a dual role, acting upstream to initiate heterochromatin silencing and downstream to stimulate a new cycle of small RNA amplification, thus establishing a self-enforcing mechanism that propagates gene silencing to future generations.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Heterocromatina/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Interferência de RNA , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismoRESUMO
The nonmuscle myosin II NMY-2 is required for cytokinesis as well as for the establishment of zygote asymmetry during embryogenesis in Caenorhabditis elegans. Here we describe two conditional nmy-2 alleles that rapidly and reversibly inactivate the protein. We show that NMY-2 has late-cell-cycle roles in maintaining embryonic asymmetries and is also required for a surprisingly late step in the maintenance of the cytokinesis furrow. Finally, during a signaling-induced asymmetric cell division, NMY-2 is required for SRC-dependent phosphotyrosine signaling and acts in parallel with WNT-signaling to specify endoderm.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Embrião não Mamífero/metabolismo , Cadeias Pesadas de Miosina/genética , Proteínas Quinases/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Padronização Corporal , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Divisão Celular , Polaridade Celular , Embrião não Mamífero/citologia , Dados de Sequência Molecular , Mutação , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Alinhamento de Sequência , Temperatura , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
In animals, Argonaute small-RNA pathways scan germline transcripts to silence self-replicating genetic elements. However, little is known about how endogenous gene expression is recognized and licensed. Here, we show that the presence of introns and, by inference, the process of mRNA splicing prevents default Argonaute-mediated silencing in the C. elegans germline. The silencing of intronless genes is initiated independently of the piRNA pathway but nevertheless engages multiple components of the downstream amplification and maintenance mechanisms that mediate transgenerational silencing, including both nuclear and cytoplasmic members of the worm-specific Argonaute gene family (WAGOs). Small RNAs amplified from intronless mRNAs can trans-silence cognate intron-containing genes. Interestingly, a second, small RNA-independent cis-acting mode of silencing also acts on intronless mRNAs. Our findings suggest that cues put in place during mRNA splicing license germline gene expression and provide evidence for a splicing-dependent and dsRNA- and piRNA-independent mechanism that can program Argonaute silencing.
Assuntos
Proteínas Argonautas/genética , Sinais (Psicologia) , Inativação Gênica/fisiologia , RNA Mensageiro/genética , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Splicing de RNA/genética , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: At the onset of embryogenesis, key developmental regulators called determinants are activated asymmetrically to specify the body axes and tissue layers. In C. elegans, this process is regulated in part by a conserved family of CCCH-type zinc finger proteins that specify the fates of early embryonic cells. The asymmetric localization of these and other determinants is regulated in early embryos through motor-dependent physical translocation as well as selective proteolysis. RESULTS: We show here that the CCCH-type zinc finger protein OMA-1 serves as a nexus for signals that regulate the transition from oogenesis to embryogenesis. While OMA-1 promotes oocyte maturation during meiosis, destruction of OMA-1 is needed during the first cell division for the initiation of ZIF-1-dependent proteolysis of cell-fate determinants. Mutations in four conserved protein kinase genes-mbk-2/Dyrk, kin-19/CK1alpha, gsk-3, and cdk-1/CDC2-cause stabilization of OMA-1 protein, and their phenotypes are partially suppressed by an oma-1 loss-of-function mutation. OMA-1 proteolysis also depends on Cyclin B3 and on a ZIF-1-independent CUL-2-based E3 ubiquitin ligase complex, as well as the CUL-2-interacting protein ZYG-11 and the Skp1-related proteins SKR-1 and SKR-2. CONCLUSIONS: Our findings suggest that a CDK1/Cyclin B3-dependent activity links OMA-1 proteolysis to completion of the first cell cycle and support a model in which OMA-1 functions to prevent the premature activation of cell-fate determinants until after they are asymmetrically partitioned during the first mitosis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/enzimologia , Proteínas de Transporte/metabolismo , Oócitos/enzimologia , Oócitos/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/metabolismo , Diferenciação Celular , Sequência Conservada , Embrião não Mamífero/citologia , Quinase 3 da Glicogênio Sintase/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Oócitos/citologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Proteínas Wnt/metabolismoRESUMO
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 , TransgenesRESUMO
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 , FenótipoRESUMO
The spindle midzone harbors both microtubules and proteins necessary for furrow formation and the completion of cytokinesis. However, the mechanisms that mediate the temporal and spatial recruitment of cell division factors to the spindle midzone and midbody remain unclear. Here we describe a mechanism governed by the conserved RNA-binding protein ATX-2/Ataxin-2, which targets and maintains ZEN-4 at the spindle midzone. ATX-2 does this by regulating the amount of PAR-5 at mitotic structures, particularly the spindle, centrosomes, and midbody. Preventing ATX-2 function leads to elevated levels of PAR-5, enhanced chromatin and centrosome localization of PAR-5-GFP, and ultimately a reduction of ZEN-4-GFP at the spindle midzone. Codepletion of ATX-2 and PAR-5 rescued the localization of ZEN-4 at the spindle midzone, indicating that ATX-2 mediates the localization of ZEN-4 upstream of PAR-5. We provide the first direct evidence that ATX-2 is necessary for cytokinesis and suggest a model in which ATX-2 facilitates the targeting of ZEN-4 to the spindle midzone by mediating the posttranscriptional regulation of PAR-5.
Assuntos
Ataxina-2/metabolismo , Ataxina-2/fisiologia , Citocinese/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Centrossomo/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fuso Acromático/metabolismoRESUMO
The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3' UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Poliadenilação/fisiologia , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismo , Animais , Caenorhabditis elegans/embriologia , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Germinativas/metabolismo , Mesoderma/metabolismo , RNA de Helmintos/genética , Proteínas de Ligação a RNARESUMO
The elegant choreography of metazoan development demands exquisite regulation of cell-division timing, orientation, and asymmetry. In this review, we discuss studies in Drosophila and C. elegans that reveal how the cell cycle machinery, comprised of cyclin-dependent kinase (CDK) and cyclins functions as a master regulator of development. We provide examples of how CDK/cyclins: (1) regulate the asymmetric localization and timely destruction of cell fate determinants; (2) couple signaling to the control of cell division orientation; and (3) maintain mitotic zones for stem cell proliferation. These studies illustrate how the core cell cycle machinery should be viewed not merely as an engine that drives the cell cycle forward, but rather as a dynamic regulator that integrates the cell-division cycle with cellular differentiation, ensuring the coherent and faithful execution of developmental programs.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Diferenciação Celular , Divisão Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Drosophila/crescimento & desenvolvimento , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proliferação de Células , Drosophila/citologia , Drosophila/metabolismo , HumanosRESUMO
Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , RNA Helicases DEAD-box/metabolismo , Interferência de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Proteínas de Caenorhabditis elegans/genética , RNA Helicases DEAD-box/genética , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Ligação a RNA/genéticaRESUMO
Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.