RESUMO
As phase separation is found in an increasing variety of biological contexts, additional challenges have arisen in understanding the underlying principles of condensate formation and function. We spoke with researchers across disciplines about their views on the ever-changing landscape of biomolecular condensates.
Assuntos
Condensados Biomoleculares , Pesquisadores , Humanos , BiologiaRESUMO
RNA granules are mesoscale assemblies that form in the absence of limiting membranes. RNA granules contain factors for RNA biogenesis and turnover and are often assumed to represent specialized compartments for RNA biochemistry. Recent evidence suggests that RNA granules assemble by phase separation of subsoluble ribonucleoprotein (RNP) complexes that partially demix from the cytoplasm or nucleoplasm. We explore the possibility that some RNA granules are nonessential condensation by-products that arise when RNP complexes exceed their solubility limit as a consequence of cellular activity, stress, or aging. We describe the use of evolutionary and mutational analyses and single-molecule techniques to distinguish functional RNA granules from "incidental condensates."
Assuntos
Grânulos Citoplasmáticos , Ribonucleoproteínas , Ribonucleoproteínas/genética , Grânulos de Ribonucleoproteínas Citoplasmáticas , RNA/químicaRESUMO
Nucleoporins (Nups) assemble nuclear pores that form the permeability barrier between nucleoplasm and cytoplasm. Nucleoporins also localize in cytoplasmic foci proposed to function as pore pre-assembly intermediates. Here, we characterize the composition and incidence of cytoplasmic Nup foci in an intact animal, C. elegans. We find that, in young non-stressed animals, Nup foci only appear in developing sperm, oocytes and embryos, tissues that express high levels of nucleoporins. The foci are condensates of highly cohesive FG repeat-containing nucleoporins (FG-Nups), which are maintained near their solubility limit in the cytoplasm by posttranslational modifications and chaperone activity. Only a minor fraction of FG-Nup molecules concentrate in Nup foci, which dissolve during M phase and are dispensable for nuclear pore assembly. Nucleoporin condensation is enhanced by stress and advancing age, and overexpression of a single FG-Nup in post-mitotic neurons is sufficient to induce ectopic condensation and organismal paralysis. We speculate that Nup foci are non-essential and potentially toxic condensates whose assembly is actively suppressed in healthy cells.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Masculino , Animais , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sêmen/metabolismo , Núcleo Celular/metabolismo , Transporte Ativo do Núcleo CelularRESUMO
In animals with germ plasm, embryonic germline precursors inherit germ granules, condensates proposed to regulate mRNAs coding for germ cell fate determinants. In Caenorhabditis elegans, mRNAs are recruited to germ granules by MEG-3, a sequence non-specific RNA-binding protein that forms stabilizing interfacial clusters on germ granules. Using fluorescence in situ hybridization, we confirmed that 441 MEG-3-bound transcripts are distributed in a pattern consistent with enrichment in germ granules. Thirteen are related to transcripts reported in germ granules in Drosophila or Nasonia. The majority, however, are low-translation maternal transcripts required for embryogenesis that are not maintained preferentially in the nascent germline. Granule enrichment raises the concentration of certain transcripts in germ plasm but is not essential to regulate mRNA translation or stability. Our findings suggest that only a minority of germ granule-associated transcripts contribute to germ cell fate in C. elegans and that the vast majority function as non-specific scaffolds for MEG-3.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Células Germinativas , Biossíntese de Proteínas , RNA Mensageiro , Proteínas de Ligação a RNA , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Células Germinativas/citologia , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ FluorescenteRESUMO
P-bodies are cytoplasmic condensates that accumulate low-translation mRNAs for temporary storage before translation or degradation. P-bodies have been best characterized in yeast and mammalian tissue culture cells. We describe here related condensates in the germline of animal models. Germline P-bodies have been reported at all stages of germline development from primordial germ cells to gametes. The activity of the universal germ cell fate regulator, Nanos, is linked to the mRNA decay function of P-bodies, and spatially-regulated condensation of P-body like condensates in embryos is required to localize mRNA regulators to primordial germ cells. In most cases, however, it is not known whether P-bodies represent functional compartments or non-functional condensation by-products that arise when ribonucleoprotein complexes saturate the cytoplasm. We speculate that the ubiquity of P-body-like condensates in germ cells reflects the strong reliance of the germline on cytoplasmic, rather than nuclear, mechanisms of gene regulation.
Assuntos
Corpos de Processamento , Proteínas de Ligação a RNA , Animais , Proteínas de Ligação a RNA/genética , Células Germinativas/metabolismo , RNA Mensageiro/genética , Regulação da Expressão Gênica , Mamíferos/genéticaRESUMO
Protein concentration gradients encode spatial information across cells and tissues and often depend on spatially localized protein synthesis. Here, we report that a different mechanism underlies the MEX-5 gradient. MEX-5 is an RNA-binding protein that becomes distributed in a cytoplasmic gradient along the anterior-to-posterior axis of the one-cell C. elegans embryo. We demonstrate that the MEX-5 gradient is a direct consequence of an underlying gradient in MEX-5 diffusivity. The MEX-5 diffusion gradient arises when the PAR-1 kinase stimulates the release of MEX-5 from slow-diffusive, RNA-containing complexes in the posterior cytoplasm. PAR-1 directly phosphorylates MEX-5 and is antagonized by the spatially uniform phosphatase PP2A. Mathematical modeling and in vivo observations demonstrate that spatially segregated phosphorylation and dephosphorylation reactions are sufficient to generate stable protein concentration gradients in the cytoplasm. The principles demonstrated here apply to any spatially segregated modification cycle that affects protein diffusion and do not require protein synthesis or degradation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Difusão , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Zigoto/química , Zigoto/metabolismoRESUMO
In animals with germ plasm, specification of the germline involves 'germ granules', cytoplasmic condensates that enrich maternal transcripts in the germline founder cells. In Caenorhabditis elegans embryos, P granules enrich maternal transcripts, but surprisingly P granules are not essential for germ cell fate specification. Here, we describe a second condensate in the C. elegans germ plasm. Like canonical P-bodies found in somatic cells, 'germline P-bodies' contain regulators of mRNA decapping and deadenylation and, in addition, the intrinsically-disordered proteins MEG-1 and MEG-2 and the TIS11-family RNA-binding protein POS-1. Embryos lacking meg-1 and meg-2 do not stabilize P-body components, misregulate POS-1 targets, mis-specify the germline founder cell and do not develop a germline. Our findings suggest that specification of the germ line involves at least two distinct condensates that independently enrich and regulate maternal mRNAs in the germline founder cells. This article has an associated 'The people behind the papers' interview.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Corpos de Processamento , Células Germinativas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular/genética , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Nuage are RNA-rich condensates that assemble around the nuclei of developing germ cells. Many proteins required for the biogenesis and function of silencing small RNAs (sRNAs) enrich in nuage, and it is often assumed that nuage is the cellular site where sRNAs are synthesized and encounter target transcripts for silencing. Using C. elegans as a model, we examine the complex multicondensate architecture of nuage and review evidence for compartmentalization of silencing pathways. We consider the possibility that nuage condensates balance the activity of competing sRNA pathways and serve to limit, rather than enhance, sRNA amplification to protect transcripts from dangerous runaway silencing.
Assuntos
Condensados Biomoleculares/química , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/química , Interferência de RNA , RNA de Helmintos/química , RNA Interferente Pequeno/química , Animais , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Condensados Biomoleculares/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Embrião não Mamífero , Grânulos de Ribonucleoproteínas de Células Germinativas/metabolismo , Grânulos de Ribonucleoproteínas de Células Germinativas/ultraestrutura , Células Germinativas/metabolismo , Células Germinativas/ultraestrutura , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
DYRKs are kinases that self-activate in vitro by autophosphorylation of a YTY motif in the kinase domain, but their regulation in vivo is not well understood. In C. elegans zygotes, MBK-2/DYRK phosphorylates oocyte proteins at the end of the meiotic divisions to promote the oocyte-to-embryo transition. Here we demonstrate that MBK-2 is under both positive and negative regulation during the transition. MBK-2 is activated during oocyte maturation by CDK-1-dependent phosphorylation of serine 68, a residue outside of the kinase domain required for full activity in vivo. The pseudotyrosine phosphatases EGG-4 and EGG-5 sequester activated MBK-2 until the meiotic divisions by binding to the YTY motif and inhibiting MBK-2's kinase activity directly, using a mixed-inhibition mechanism that does not involve tyrosine dephosphorylation. Our findings link cell-cycle progression to MBK-2/DYRK activation and the oocyte-to-embryo transition.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Proteínas Tirosina Quinases/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Humanos , Oócitos/metabolismoRESUMO
Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.
Assuntos
Sistemas CRISPR-Cas , Inativação Gênica , Modelos Animais , Morfolinos/farmacologia , Mutagênese , Mutação/genética , Interferência de RNA , Animais , Genótipo , Humanos , Fenótipo , Especificidade da EspécieRESUMO
The MARK/PAR-1 family of kinases are conserved regulators of cell polarity that share a conserved C-terminal kinase-associated domain (KA1). Localization of MARK/PAR-1 kinases to specific regions of the cell cortex is a hallmark of polarized cells. In Caenorhabditiselegans zygotes, PAR-1 localizes to the posterior cortex under the influence of another polarity kinase, aPKC/PKC-3. Here, we report that asymmetric localization of PAR-1 protein is not essential, and that PAR-1 kinase activity is regulated spatially. We find that, as in human MARK1, the PAR-1 KA1 domain is an auto-inhibitory domain that suppresses kinase activity. Auto-inhibition by the KA1 domain functions in parallel with phosphorylation by PKC-3 to suppress PAR-1 activity in the anterior cytoplasm. The KA1 domain also plays an additional role that is essential for germ plasm maintenance and fertility. Our findings suggest that modular regulation of kinase activity by redundant inhibitory inputs contributes to robust symmetry breaking by MARK/PAR-1 kinases in diverse cell types.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Polaridade Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Serina-Treonina Quinases/fisiologia , Alelos , Animais , Linhagem da Célula , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Humanos , Microscopia Confocal , Mutação , Fosforilação , Domínios Proteicos , Proteína Quinase C/fisiologia , Interferência de RNA , Treonina/químicaRESUMO
The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only â¼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA , Endonucleases/metabolismo , Edição de Genes/métodos , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Células HEK293 , Humanos , Camundongos , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido NucleicoRESUMO
The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes , Técnicas de Transferência de Genes , RNA Guia de Cinetoplastídeos/genética , Alelos , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR , Caenorhabditis elegans/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Embrião não Mamífero , Endonucleases/metabolismo , Marcação de Genes/métodos , Genoma , Microinjeções , Mutação , RNA Guia de Cinetoplastídeos/metabolismo , Reparo de DNA por RecombinaçãoRESUMO
Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (â¼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/genética , DNA Recombinante/genética , Edição de Genes/métodos , Recombinação Homóloga/genética , Alelos , Animais , Proteínas Associadas a CRISPR/genética , Quebras de DNA de Cadeia Dupla , Conversão Gênica/genética , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Reparo de DNA por Recombinação/genética , Moldes GenéticosRESUMO
In the C. elegans germline, maintenance of undifferentiated stem cells depends on the PUF family RNA-binding proteins FBF-1 and FBF-2. FBF-1 and FBF-2 are 89% identical and are required redundantly to silence the expression of mRNAs that promote meiosis. Here we show that, despite their extensive sequence similarity, FBF-1 and FBF-2 have different effects on target mRNAs. FBF-1 promotes the degradation and/or transport of meiotic mRNAs out of the stem cell region, whereas FBF-2 prevents translation. FBF-2 activity depends on the P granule component PGL-1. PGL-1 is required to localize FBF-2 to perinuclear P granules and for efficient binding of FBF-2 to its mRNA targets. We conclude that multiple regulatory mechanisms converge on meiotic RNAs to ensure silencing in germline stem cells. Our findings also support the view that P granules facilitate mRNA silencing by providing an environment in which translational repressors can encounter their mRNA targets immediately upon exit from the nucleus.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Células-Tronco Embrionárias/metabolismo , Células Germinativas/metabolismo , Interferência de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Meiose/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genéticaRESUMO
The KT3 antibody is a commercially available antibody that recognizes the P granule protein PGL-3 (Takeda et al., 2008). Using immunostaining and western blotting of purified peptide fragments, we show that KT3 recognizes both PGL-3 and its paralog PGL-1 , likely through a shared epitope in the intrinsically disordered region.
RESUMO
C. elegans P granules are conserved cytoplasmic ribonucleoprotein complexes that are unique to the germline and essential for fertility. During most of germline development, P granules are perinuclear and associate with clusters of nuclear pores. In an RNAi screen against nucleoporins, we have identified a specific nucleoporin essential for P granule integrity and function. The C. elegans homolog of vertebrate Nup98 (CeNup98) is enriched in P granules and associates with the translationally repressed, P granule-enriched mRNA nos-2 (nanos homolog). Loss of CeNup98 causes P granules to disperse in the cytoplasm and to release nos-2 mRNA. Embryos depleted for CeNup98 express a nos-2 3'UTR reporter prematurely. In the mouse, Nup98 immunoprecipitates with the germ granule component MVH. Our findings suggest that, in germ cells, the function of Nup98 extends beyond transport at the nuclear pore to include mRNA regulation in the cytoplasm.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Células Germinativas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Embrião não Mamífero/metabolismo , Imunoprecipitação , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
FBF-1 and FBF-2 (collectively FBF) are two nearly identical Puf-domain RNA-binding proteins that regulate the switch from mitosis to meiosis in the C. elegans germline. In germline stem cells, FBF prevents premature meiotic entry by inhibiting the expression of meiotic regulators, such as the RNA-binding protein GLD-1. Here, we demonstrate that FBF also directly inhibits the expression of structural components of meiotic chromosomes. HIM-3, HTP-1, HTP-2, SYP-2 and SYP-3 are components of the synaptonemal complex (SC) that forms between homologous chromosomes during meiotic prophase. In wild-type germlines, the five SC proteins are expressed shortly before meiotic entry. This pattern depends on FBF binding sites in the 3' UTRs of the SC mRNAs. In the absence of FBF or the FBF binding sites, SC proteins are expressed precociously in germline stem cells and their precursors. SC proteins aggregate and SC formation fails at meiotic entry. Precocious SC protein expression is observed even when meiotic entry is delayed in fbf mutants by reducing GLD-1. We propose that parallel regulation by FBF ensures that in wild-type gonads, meiotic entry is coordinated with just-in-time synthesis of synaptonemal proteins.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células-Tronco/metabolismo , Complexo Sinaptonêmico/metabolismo , Regiões 3' não Traduzidas , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Regulação para Baixo , Inativação Gênica , Células Germinativas/citologia , Meiose , Camundongos , Mitose , Mutação , Proteínas Nucleares/genética , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Células-Tronco/citologiaRESUMO
Polarization of the C. elegans zygote is initiated by ECT-2-dependent cortical flows, which mobilize the anterior PAR proteins (PAR-3, PAR-6 and PKC-3) away from the future posterior end of the embryo marked by the sperm centrosome. Here, we demonstrate the existence of a second, parallel and redundant pathway that can polarize the zygote in the absence of ECT-2-dependent cortical flows. This second pathway depends on the polarity protein PAR-2. We show that PAR-2 localizes to the cortex nearest the sperm centrosome even in the absence of cortical flows. Once on the cortex, PAR-2 antagonizes PAR-3-dependent recruitment of myosin, creating myosin flows that transport the anterior PAR complex away from PAR-2 in a positive-feedback loop. We propose that polarity in the C. elegans zygote is initiated by redundant ECT-2- and PAR-2-dependent mechanisms that lower PAR-3 levels locally, triggering a positive-feedback loop that polarizes the entire cortex.