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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 RNAbinding 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. [ABSTRACT FROM AUTHOR]

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 RNAbinding 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. [ABSTRACT FROM AUTHOR]

Membraneless organelles are RNA–protein assemblies which have been implicated in post‐transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain‐containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi‐domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule. [ABSTRACT FROM AUTHOR]

RNA silencing pathways are complex, highly conserved, and perform crucial regulatory roles. In Caenorhabditis elegans germlines, RNA surveillance occurs through a series of perinuclear germ granule compartments - P granules, Z granules, SIMR foci, and Mutator foci - multiple of which form via phase separation. Although the functions of individual germ granule proteins have been extensively studied, the relationships between germ granule compartments (collectively, 'nuage') are less understood. We find that key germ granule proteins assemble into separate but adjacent condensates, and that boundaries between germ granule compartments re-establish after perturbation. We discover a toroidal P granule morphology, which encircles the other germ granule compartments in a consistent exterior-to-interior spatial organization, providing broad implications for the trajectory of an RNA as it exits the nucleus. Moreover, we quantify the stoichiometric relationships between germ granule compartments and RNA to reveal discrete populations of nuage that assemble in a hierarchical manner and differentially associate with RNAi-targeted transcripts, possibly suggesting functional differences between nuage configurations. Our work creates a more accurate model of C. elegans nuage and informs the conceptualization of RNA silencing through the germ granule compartments. [ABSTRACT FROM AUTHOR]

The copackaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to posttranscriptionally regulate germline mRNAs. In Drosophila melanogaster , mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3′ untranslated region (UTR) of germ granule mRNAs. Interestingly, the 3′ UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species and we hypothesized that this diversity influences homotypic clustering. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, we discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species' homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, we found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos , pgc , osk levels and/or homotypic clustering efficacy. Furthermore, we demonstrated how the nos 3′ UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. Our results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, many macromolecules are organized as membraneless biomolecular condensates (or biocondensates). Liquid–liquid and liquid–solid phase transitions are the drivers of the condensation process. The absence of membrane borders makes biocondensates very flexible in their composition and functions, which vary in different cells and tissues. Some biocondensates are specific for germ line cells and are, thus, termed germ granules. This review summarizes the recent data on the composition of germ granules and their functions in gametes. According to these data, germ granules are involved in the determination of germline cells in some animals, such as Amphibia. In other animals, such as Mammalia, germ granules are involved in the processes of transposons inactivation and sequestration of mRNA and proteins to temporarily decrease their activity. The new data on germ granules composition and functions sheds light on germ cell differentiation and maturation properties. [ABSTRACT FROM AUTHOR]

Animal development is dictated by the selective and timely decay of mRNAs in developmental transitions, but the impact of mRNA decapping scaffold proteins in development is unclear. This study unveils the roles and interactions of the DCAP-2 decapping scaffolds EDC-3 and EDC-4 in the embryonic development of C. elegans. EDC-3 facilitates the timely removal of specific embryonic mRNAs, including cgh-1 , car-1 , and ifet-1 by reducing their expression and preventing excessive accumulation of DCAP-2 condensates in somatic cells. We further uncover a role for EDC-3 in defining the boundaries between P bodies, germ granules, and stress granules. Finally, we show that EDC-4 counteracts EDC-3 and engenders the assembly of DCAP-2 with the GID (CTLH) complex, a ubiquitin ligase involved in maternal-to-zygotic transition (MZT). Our findings support a model where multiple RNA decay mechanisms temporally clear maternal and zygotic mRNAs throughout embryonic development. [Display omitted] • EDC-3 restricts and EDC-4 promotes DCAP-2/P body condensate assembly • EDC-3 promotes clearance of decapping scaffold mRNAs in the germline and soma • EDC-3 prevents aberrant scaffolding of condensate proteins on DCAP-2 • EDC-4 facilitates DCAP-2 interaction with the GID (CTLH) complex The timely decay of mRNAs is essential for the embryonic development of all metazoans. Vidya et al. detail how two decapping scaffold proteins (EDC-3 and EDC-4) distinctly regulate the localization and interactions of DCAP-2, an enzyme that irreversibly catalyzes mRNA decay, into cytoplasmic condensates throughout C. elegans embryonic development. [ABSTRACT FROM AUTHOR]

Cells spatially organize their molecular components to carry out fundamental biological processes and guide proper development. The spatial organization of RNA within the cell can both promote and result from gene expression regulatory control. Recent studies have demonstrated diverse associations between RNA spatial patterning and translation regulatory control. One form of patterning, compartmentalization in biomolecular condensates, has been of particular interest. Generally, transcripts associated with cytoplasmic biomolecular condensates—such as germ granules, stress granules, and P-bodies—are linked with low translational status. However, recent studies have identified new biomolecular condensates with diverse roles associated with active translation. This review outlines RNA compartmentalization in various condensates that occur in association with repressed or active translational states, highlights recent findings in well-studied condensates, and explores novel condensate behaviors. [ABSTRACT FROM AUTHOR]

Tandemly repeated pericentromeric noncoding DNA (TR DNA) makes up approximately 10% of the human genome. Pericentromeric TR DNA includes classical human satellites 1, 2, 3 (HS1, HS2, HS3), which are transcribed in somatic cells. We have previously shown the presence of HS2/HS3 transcripts in late human oogenesis and sequenced them. It has been suggested that the RNPs found may be the site of spatial sequestration of RNA and proteins at the end of human oocytes maturation. The aim of this work was to develop a method for inactivating HS2/HS3 transcripts using antisense oligonucleotides to assess its effect on the size and quantity of DDX4-containing RNPs in late human oogenesis. Inactivation of HS2/HS3 transcription at the end of human oocytes maturation by the microinjection method resulted in a significant decrease of the total HS2/HS3 RNA signal, detected by the method of fluorescence in situ hybridization (FISH). At the same time, an increase in the number of inclusions positive to antibodies to RNA helicase DDX4 was observed. Supposedly, upon inactivation of HS2/HS3 transcription, dissociation of DDX4-containing RNP particles occurred. Such changes of the RNP particles can play a critical role in the development of oocytes and be a cause of the arrest of maturation or the occurrence of pathological syndromes, including those associated with problems of fertilization. [ABSTRACT FROM AUTHOR]

Many morphogenetic processes during ontogenesis are determined by changes in the structure and functions of embryonic cells. The greatest plasticity of the cellular organization is given by membrane-less organelles or biomolecular condensates, which can be formed in the nucleus or cytoplasm by the mechanism of liquid–liquid phase separation. The flexibility of biogenesis of biomolecular condensates and the high dynamics of their components make it possible to change the cellular organization quickly, which leads to changes in the fate of cells, and, as a result, in the course of embryogenesis. In this review, using the example of germ granules, one of the types of membrane-less structures, the relationship between the plasticity of cellular organization and the implementation of early embryogenesis processes is discussed. [ABSTRACT FROM AUTHOR]

Germ granules are biomolecular condensates that form in germ cells of all/most animals, where they regulate mRNA expression to promote germ cell function and totipotency. In the adult Caenorhabditis elegans germ cell, these granules are composed of at least four distinct sub‐compartments, one of which is the Z granule. To better understand the role of the Z granule in germ cell biology, we conducted a genetic screen for genes specifically required for Z granule assembly or morphology. Here, we show that zsp‐1, which encodes a low‐complexity/polyampholyte‐domain protein, is required for Z granule homeostasis. ZSP‐1 localizes to the outer surface of Z granules. In the absence of ZSP‐1, Z granules swell to an abnormal size, fail to segregate with germline blastomeres during development, and lose their liquid‐like character. Finally, ZSP‐1 promotes piRNA‐ and siRNA‐directed gene regulation and germline immortality. Our data suggest that Z granules coordinate small RNA‐based gene regulation to promote germ cell function and that ZSP‐1 helps/is need to maintain Z granule morphology and liquidity. SYNOPSIS: In Caenorhabditis elegans, the Z granule is one of at least four proposed sub‐compartments of the germline perinuclear nuage, which regulates mRNA expression for germ cell function and totipotency. Here, ZSP‐1 is identified as a regulator of Z granules homeostasis. The low‐complexity/polyampholyte‐domain protein ZSP‐1 localizes to the surface of Z granules.Z granule homeostasis requires ZSP‐1, which regulates their morphology and viscosity.ZSP‐1 promotes germline small RNA pathways and germline immortality. [ABSTRACT FROM AUTHOR]

Germ granules are hallmarks of all germ cells. Early ultrastructural studies in Drosophila first described these membraneless granules in the oocyte and early embryo as filled with amorphous to fibrillar material mixed with RNA. Genetic studies identified key protein components and specific mRNAs that regulate germ cell‐specific functions. More recently these ultrastructural studies have been complemented by biophysical analysis describing germ granules as phase‐transitioned condensates. In this review, we provide an overview that connects the composition of germ granules with their function in controlling germ cell specification, formation and migration, and illuminate these mysterious condensates as the gatekeepers of the next generation. [ABSTRACT FROM AUTHOR]

Drosophila Me31B is a conserved protein of germ granules, ribonucleoprotein complexes essential for germ cell development. Me31B post-transcriptionally regulates mRNAs by interacting with other germ granule proteins. However, a Me31B interactome is lacking. Here, we use an in vivo proteomics approach to show that the Me31B interactome contains polypeptides from four functional groups: RNA regulatory proteins, glycolytic enzymes, cytoskeleton/motor proteins, and germ plasm components. We further show that Me31B likely colocalizes with the germ plasm components Tudor (Tud), Vasa, and Aubergine in the nuage and germ plasm and provide evidence that Me31B may directly bind to Tud in a symmetrically dimethylated arginine-dependent manner. Our study supports the role of Me31B in RNA regulation and suggests its novel roles in germ granule assembly and function. [ABSTRACT FROM AUTHOR]

RNA granules are membraneless condensates that provide functional compartmentalization within cells. The mechanisms by which RNA granules form are under intense investigation. Here, we characterize the role of mRNAs and proteins in the formation of germ granules in Drosophila. Super-resolution microscopy reveals that the number, size, and distribution of germ granules is precisely controlled. Surprisingly, germ granule mRNAs are not required for the nucleation or the persistence of germ granules but instead control their size and composition. Using an RNAi screen, we determine that RNA regulators, helicases, and mitochondrial proteins regulate germ granule number and size, while the proteins of the endoplasmic reticulum, nuclear pore complex, and cytoskeleton control their distribution. Therefore, the protein-driven formation of Drosophila germ granules is mechanistically distinct from the RNA-dependent condensation observed for other RNA granules such as stress granules and P-bodies. [Display omitted] • The number, size, and distribution of germ granules is precisely regulated • Oskar germ granules do not require RNA for their nucleation and stability • RNA regulators and mitochondrial proteins control germ granule number and size • ER, nuclear pore, and cytoskeletal proteins regulate germ granule distribution Curnutte et al. observe that germ granule proteins rather than mRNAs promote the nucleation and stability of Oskar germ granules in Drosophila. While RNA regulators and mitochondrial proteins control the number and size of germ granules, the proteins of the ER, nuclear pores, and cytoskeleton control their distribution. [ABSTRACT FROM AUTHOR]

Macromolecular complexes and organelles play crucial roles within cells, but their native architectures are often unknown. Here, we use an evolutionarily conserved germline organelle, the germ granule, as a paradigm. In Drosophila embryos, we map one of its interactomes using a novel in vivo crosslinking approach that employs two interacting granule proteins and determines their common neighbor molecules. We identified an in vivo granule assembly of Tudor, Aubergine, motor and metabolic proteins, and RNA helicases, and provide evidence for direct interactions within this assembly using purified components. Our study indicates that germ granules contain efficient biochemical reactors involved in post-transcriptional gene regulation. [ABSTRACT FROM AUTHOR]

Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. [ABSTRACT FROM AUTHOR]

Large cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation. [ABSTRACT FROM AUTHOR]

RNA granules are ubiquitous. Composed of RNA-binding proteins and RNAs, they provide functional compartmentalization within cells. They are inextricably linked with RNA biology and as such are often referred to as the hubs for post-transcriptional regulation. Much of the attention has been given to the proteins that form these condensates and thus many fundamental questions about the biology of RNA granules remain poorly understood: How and which RNAs enrich in RNA granules, how are transcripts regulated in them, and how do granule-enriched mRNAs shape the biology of a cell? In this review, we discuss the imaging, genetic, and biochemical data, which have revealed that some aspects of the RNA biology within granules are carried out by the RNA itself rather than the granule proteins. Interestingly, the RNA structure has emerged as an important feature in the post-transcriptional control of granule transcripts. This review is part of the Special Issue in the Frontiers in RNA structure in the journal Molecules. [ABSTRACT FROM AUTHOR]

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs). [ABSTRACT FROM AUTHOR]