P-granules are conserved cytoplasmic organelles, similar to nuage, that are present in Caenorhabditis elegans germ cells. Based on the prevailing sterility phenotype of the component mutants, P-granules have been seen as regulators of germ cell development and function. Yet, specific germline defects resulting from P-granule failure vary, depending on which component(s) are inactivated, at which stage of development, as well as on the presence of stress factors during animal culture. This review discusses the unifying themes in many P-granule functions, with the main focus on their role as organizing centers nucleating RNA regulation in the germ cell cytoplasm. Mol. Reprod. Dev. 80:624–631, 2013. © 2012 Wiley Periodicals, Inc.
primordial germ cell
Year 2012 marks the 30th anniversary of the discovery of cytoplasmic granules unique to the germline cells in the nematode Caenorhabditis elegans (Strome and Wood, 1982). These were termed “P-granules” after the P blastomere embryonic lineage that gives rise to the germ cell precursor, and eventually to all germ cells of the animal. The germline lineage is set aside early in C. elegans development (Fig. 1; see also Pazdernik and Schedl, 2013). The P-lineage blastomere divides only four times to form the germline progenitor cell, P4. P4 divides once during embryogenesis, forming Z2 and Z3, which stay quiescent until the end of the first larval stage. Late P4, Z2, and Z3 cells are considered primordial germ cells (PGCs) of the C. elegans embryo. Mitotic germ cell proliferation at the end of L1 and through the L2 stage is followed first by meiotic differentiation of a subpopulation of germ cells at the L3 stage, then by spermatogenesis at L4, and finally by a switch to oogenesis in the adult.
P-granules are recognized at every stage of germ cell development, except that of mature sperm (Strome and Wood, 1982). P-granules are perinuclear for the majority of the life cycle, dissociating into the cytoplasm in the oocyte, and remaining cytoplasmic up to the P3 cell stage (Figs. 1 and 2). Ultrastructural analysis of P-granules described their characteristic electron-dense fibrillar organization, similar to the germ granules or nuage present in other animal species (Wolf et al., 1983). Despite the lack of a membrane boundary, P-granules appear to be well-delineated from the surrounding cytoplasm. Since their first discovery, a large number of P-granule components have been identified (Updike and Strome, 2010). Genetic studies of P-granule components suggest that P-granules are intimately associated with germ cell fate and function since the mutations in the components' genes frequently cause sterility. This review will discuss the state of our knowledge about P-granule components, their function in the germ cell's lifecycle, and several recent hypotheses for the molecular mechanisms of P-granule's function.
The constitutive components of P-granules are found in P-granules at every stage of development, thus molecularly defining the organelle. One constitutive component family belongs to the nematode homologs of the Drosophila DEAD-box helicase Vasa (Hay et al., 1988a, 1988b; Lasko and Ashburner, 1988), one of the first described germ granule components and one conserved in all animal phyla (reviewed in Yajima and Wessel, 2011). This germline helicase (GLH) family consists of GLH-1 through 4, each of which has a DEAD-box RNA helicase motif and varying domains at the N-terminus (Roussell and Bennett, 1993; Gruidl et al., 1996; Kuznicki et al., 2000). The other prominent, core component family is comprised of PGL-1 and PGL-3, which are Caenorhabditis-specific RGG-domain proteins predicted to bind RNA (Kawasaki et al., 2004). PGL and GLH proteins are critical for maintaining P-granule integrity and are mutually required for aggregation into foci (Gruidl et al., 1996; Kawasaki et al., 1998; Kuznicki et al., 2000; Spike et al., 2008a; Hanazawa et al., 2011). Localization of PGL-1 to perinuclear foci is lost in the glh-1;glh-4 mutant gonads. Similarly, in pgl-1;pgl-3 mutants, P-granule organization of GLH-1 is lost. Another structurally important, constitutive P-granule component is the worm-specific protein DEPS-1 (Spike et al., 2008b). DEPS-1 function is required for PGL-1 and PGL-3 assembly into P-granules. Mutants in deps-1, pgl, and glh genes interfere with germ cell proliferation and differentiation, leading to worm sterility. The most severe phenotypes are observed when single-mutant worms are grown at high temperature (26°C), or at the regular culture temperature (20°C) when multiple P-granule components are compromised.
The presence of a core P-granule structure recruits many transient components to these organelles. Transient components are enriched in P-granules, but localize to P-granules only during a particular period in development. Examples of transient components of P-granules are PIE-1, which is present only during the beginning of embryogenesis (Mello et al., 1996), and PAN-1, which is missing from embryonic P-granules and localizes to P-granules only in the larval and adult germ cells (Gao et al., 2012). Additionally, some P-granule components are also found outside P-granules or even in somatic cells. One of these is a nuclear pore component NPP-10, a homolog of the vertebrate nucleoporin Nup98, which is found at the nuclear envelope in all somatic and germ cells in addition to contributing to P-granules in germ cells (Voronina and Seydoux, 2010). A comprehensive list including most transient components was recently published (Updike and Strome, 2010).
The majority of the currently identified protein components of P-granules possess RNA-binding domains (Updike and Strome, 2010); this may not be surprising as the presence of RNA in P-granules has been long expected and documented. P-granule-enriched RNAs are polyadenylated and contain the SL1 trans-spliced leader sequence, suggesting that they are mRNAs (Seydoux and Fire, 1994), but only six specific mRNA species have yet been reported to be enriched in P-granules. These include the nanos homolog nos-2 mRNA in P-granules of the embryonic germ cells and pos-1, skn-1, par-3, mex-1, gld-1, and nos-2 in the adult germline P-granules (Subramaniam and Seydoux, 1999; Schisa et al., 2001). Additionally, a number of P-granule components are involved in the biosynthesis or function of small RNAs, including Dicer homolog DCR-1, Dicer-related helicase DRH-1, RNA-dependent RNA polymerase EGO-1, and Argonaute proteins CSR-1, PRG-1, and WAGO-1 (Batista et al., 2008; Claycomb et al., 2009; Gu et al., 2009; Beshore et al., 2011). It is likely that P-granule-enriched Argonautes are in complex with their cognate small RNAs; however, so far no subcellular localization of small RNA has been reported in C. elegans. A clear next challenge of the field is to identify the full RNA repertoire of the P-granules (including mRNAs, small RNAs, and any non-coding RNAs).
The absence of a surrounding membrane permits the free exchange of P-granule components with the surrounding cytoplasm. P-granule stability and dynamics greatly depend on the developmental stage of the germ cell. In the meiotic cells of the adult germline, the perinuclear P-granules possess a stable, underlying structure supporting their position and shape for up to 40 min of live-imaging observations, but still permitting the rapid exchange of the fluorescently tagged components (∼15 sec half-time; Sheth et al., 2010). In contrast, oocyte P-granules are more labile, can be displaced from the nucleus by shear stress, and exhibit “dripping” and “wetting” behaviors prompting a “liquid droplet” analogy (Brangwynne et al., 2009). The dynamic nature of P-granules is most pronounced in the single-cell embryo, where cytoplasmic P-granules exhibit shrinking and growing behaviors, depending on the cell cycle stage and relative position along the anterior-posterior axis (Brangwynne et al., 2009; Gallo et al., 2010). Regulation of P-granule dynamics happens at the post-translational level: in embryonic cells, it is affected by the protein regulators promoting either P-granule disassembly, such as MEX-5, or P-granule stabilization, such as PAR-1 kinase and PP2A phosphatase (Cheeks et al., 2004; Brangwynne et al., 2009; Gallo et al., 2010).
FUNCTIONS IN DEVELOPMENT
Germ Cell Specification
P-granule localization to germ cell precursors during embryogenesis led to a hypothesis that selective inheritance of P-granules directs germ cell precursor cell fate specification, similar to Drosophila polar granules. The simple model that P-granules are sufficient to confer germ cell fate has been challenged, however. The first line of conflicting evidence suggests that ectopic expression of P-granule components or their mis-partitioning to somatic tissues of the larvae does not induce supernumerary germ cells (Strome et al., 1995; Unhavaithaya et al., 2002; Wang et al., 2005; Petrella et al., 2011). This inability to direct tissue specification may result from ectopic expression of a subset of P-granule components or from the fact that, in most cases, this ectopic expression is not detected until later in development, when the affected somatic cells have committed to their terminal differentiation fate. Additionally, the described mutations disrupt general transcriptional regulators expected to broadly affect transcriptional status of the cells (retinoblastoma pathway, chromatin modifiers), thus confounding the issue of exactly which genes are misexpressed in each case and what overall environment P-granule components become a part of.
The second line of evidence argues that disrupting normal accumulation of P-granule material in the germline lineage does not prevent germ cell specification (Gallo et al., 2010). PPTR-1 is a regulatory subunit of PP2A phosphatase that directs substrate specificity of the PP2A heterotrimeric complex. In pptr-1 mutants, P-granules completely disperse during mitotic divisions, resulting in equal segregation of P-granule components between the daughter cells and a corresponding decrease in the amount of P-granule material in germ cell precursors. Surprisingly, the transient P-granule component PIE-1 still segregates normally. The pptr-1 mutant worms are fertile at the permissive temperature of 20°C, suggesting that germ cells are specified and function normally and that no supernumerary germ cells form during embryogenesis. At 26°C, 20% of pptr-1 mutant worms have underproliferated germlines, reminiscent of a mutant phenotype of P-granule components. Indeed, this phenotype is exacerbated by pgl-1 mutation, as 15% of pgl-1;pptr-1 mutants are sterile even at the permissive temperature (Gallo et al., 2010). Since the pptr-1 mutation does not completely eliminate P-granule components in the developing germ cells, the possibility remains that P-granules contribute to germ cell specification, but do not instruct germ cell fate.
Germ Cell Survival
Sterility of several P-granule component mutants results from death or degeneration of germ cells. Homologs of a conserved germ cell development regulator Nanos, nos-1 and nos-2, are redundantly required for germ cell survival (Subramaniam and Seydoux, 1999). In worms lacking function of both nos-1 and nos-2, germ cells withdraw from proliferation, disperse their P-granules, and degenerate at the L3 developmental stage. Interestingly, a similar phenotype is observed in worms mutant for another paralogous pair of P-granule component genes, meg-1 and meg-2 (Leacock and Reinke, 2008). MEG-1 and MEG-2 are partially redundant, novel proteins transiently associating with P-granules in P2 to P4 germ cells. Similar mutant phenotypes may indicate that nos and meg genes contribute to the same regulatory process, and indeed meg-1 and nos-2 genetically interact; the double meg-1;nos-2 mutant has a more severe phenotype that each of the single mutants (Kapelle and Reinke, 2011). Z2 and Z3 PGCs in the meg-1;nos-2 embryos never proliferate, disperse perinuclear P-granules into the cytoplasm, and die by the first larval stage. Germ cell death in this class of mutants is carried out by a combined effect of apoptosis and an independent “death program” (Subramaniam and Seydoux, 1999; Kapelle and Reinke, 2011). Although the germ cells in these mutants are recognizable by the localization of maternally inherited P-granule components, it is not clear if the zygotic program of germ cell specification is fully intact.
Adult Gamete Formation
Sterility of the P-granule-component mutant worms is associated with multiple defects in gonadal development, the most consistent of which is an underproliferated germline containing about one-third of the wild-type number of germ cell nuclei and lacking molecular markers of mitotic cells such as phosphorylated Histone H3 Serine 10 (Gruidl et al., 1996; Kuznicki et al., 2000; Kawasaki et al., 2004; Spike et al., 2008a, 2008b). Another prominent defect in the P-granule component mutants is the lack of differentiated gametes. Depending on the specific gene(s) inactivated and the presence of temperature stress, the phenotypes range from delayed and defective spermatogenesis and a lack of oocytes, to a failure of spermatogenesis while the oocytes are still produced, to a lack of gametes altogether (Kuznicki et al., 2000; Kawasaki et al., 2004; Spike et al., 2008a, 2008b). The mechanisms underlying such variable temperature-sensitive phenotypes are not understood, and present an exciting new avenue of study.
With the comprehensive understanding of the developmental requirements for P-granules, the next big goal is to identify the cellular and molecular mechanisms behind diverse P-granule functions in germ cells. Since P-granules are enriched in RNA-binding proteins and appear to contain some RNAs, one hypothesis is that they are required to regulate levels or the translational activity of germ cells mRNAs.
MOLECULAR MECHANISM: RNA REGULATION
One characteristic feature of germ cells is their use of post-transcriptional gene regulation as the dominant mechanism to control gene expression (Merritt et al., 2008). Germ cells produce and accumulate multiple mRNA species that are stored in a repressed state for varying periods of time, and are then activated upon specific developmental transitions. This post-transcriptional regulation is carried out by sequence-specific RNA-binding proteins (Kimble, 2011; Sengupta and Boag, 2012). Many translational regulator proteins that function in the germline are transient components of P-granules, and recent evidence suggests that at least some of them require P-granule integrity for their efficient function. One benefit of gathering mRNAs and RNA regulators into cytoplasmic foci is the possibility of better control of mRNA partitioning among regulatory complexes.
Perinuclearly localized P-granules overlap clusters of nuclear pores in adult and embryonic germ cells, effectively covering at least 75% of the available nuclear pores on most germ cell nuclei in the gonad (Pitt et al., 2000). Similar to the proteins that comprise the nuclear pores, P-granules contain multiple FG-repeat proteins and can block passive diffusion of molecules above 40 kDa (Sheth et al., 2010; Voronina and Seydoux, 2010; Updike et al., 2011). As P-granules are overlaying the nuclear pores, the majority of mRNA molecules exported from germ cell nuclei is likely to transit through P-granules. During the developmental stages where P-granules are cytoplasmic, germ cell mRNA transcription is shut off. Perinuclear localization of P-granules during the stages of active germ cell transcription is a perfect location for continuous surveillance of the germ cell's transcriptome. Accordingly, P-granules have been shown to transiently intercept nascent mRNAs in adult germ cells (Sheth et al., 2010). By trapping mRNAs en route to the cytoplasm, P-granules may provide a secluded environment where an mRNA can effectively form a complex with its developmental regulators, protected from the competition of the cytoplasmic translational machinery. Such mechanism could promote effective control of the post-transcriptional fate of germline mRNAs.
Post-transcriptional regulation can be carried out at multiple steps of an mRNA's life, one of which is regulation of transcript stability. During the pachytene stage of oogenesis, germ cells synthesize mRNAs that are inherited by developing oocytes and forming embryos (Schisa et al., 2001). Many of these maternal mRNAs are complexed with the transient components of P-granules, CGH-1 and GLD-1 (Boag et al., 2008; Scheckel et al., 2012). Loss of CGH-1 as well as GLD-1 leads to a decrease in the levels of the target mRNAs that are able to accumulate in germline (Boag et al., 2008; Noble et al., 2008; Scheckel et al., 2012). Many of CGH-1 and GLD-1 targets overlap, and are stabilized independently by each of these proteins such that the defects in mRNA accumulation are greatest in the double gld-1;cgh-1 mutant (Scheckel et al., 2012).
Accumulation of maternal mRNAs in germ cells in advance of the time when they are required in embryonic development means that these mRNAs need to be stored in a silenced state. An example of such maternal mRNA stored for an extended period is the Nanos homolog nos-2, a critical regulator of the germ cell fate. nos-2 is transcribed by pachytene germ cells, stored in an inactive form through oogenesis, and nos-2 mRNA is then inherited by the P-lineage blastomeres. The germ cell precursor blastomeres give rise to several somatic lineages before exclusively committing to the germline fate, making it necessary to silence nos-2 mRNA until this final commitment (Subramaniam and Seydoux, 1999; D'Agostino et al., 2006). Translational activity of nos-2 mRNA in the embryo is regulated in a combinatorial fashion by multiple protein factors: MEX-3 and SPN-4 function as translational repressors, while POS-1 counteracts their influence to activate nos-2 translation in the PGC (Jadhav et al., 2008). All these proteins are transient components of P-granules (Draper et al., 1996; Tabara et al., 1999; Ogura et al., 2003), and nos-2 mRNA becomes especially enriched in P-granules in P3 cells, where it is normally repressed (Fig. 3A, Gallo et al., 2008; Voronina and Seydoux, 2010). This raises the question of whether or not P-granule structure is important for the faithful regulation of nos-2 mRNA activity. Indeed, upon acute dispersal of P-granules by depletion of P-granule-enriched nucleoporin NPP-10/Nup98, nos-2 mRNA is largely released from residual P-granules and translational repression is lost in 40–60% of the treated embryos (Voronina and Seydoux, 2010).
In the adult C. elegans germline, cells in the distal region of the gonad comprise the stem cell population. The stem cells sustain growth of the germline during the larval stages and replenish gametes in the adult in response to the signaling from the stem cell niche (reviewed in Hansen and Schedl, 2013). As the distal cells move out of the stem cell region, they start accumulating meiotic mRNAs, which are kept silent by the combined action of FBF-1 and FBF-2, two PUF-domain translational repressors, until the transition to meiosis (Kimble, 2011). Recent evidence suggests that FBF-2, but not FBF-1, is a transient component of P-granules (Voronina et al., 2012). FBF-2 is enriched in perinuclear foci coincident with P-granules, as defined by PGL-1, and is also diffusely distributed in the cytoplasm. In both the pgl-1 mutant and after glh-1(RNAi), each of which results in dispersal of perinuclear PGL-1, the perinuclear enrichment of FBF-2 is lost. In contrast, FBF-1 is localized to perinuclear foci adjacent, but rarely overlapping, with P-granules, and this localization does not depend on pgl-1 or glh-1. In the pgl-1 mutant or after glh-1(RNAi), the repressive capability of FBF-2 is compromised, which is at least in part due to the less effective binding of FBF-2 to its target mRNAs (Voronina et al., 2012). Thus FBF-2 target mRNAs, which are maintained in the repressed state outside of P-granules, require the presence of P-granule structures to ensure efficient formation of repressive FBF-2/mRNA complexes. P-granules could provide this function by recruiting FBF-2 to the sites of mRNA export (Fig. 3B). The major difference between the scenarios presented in Figure 3A versus 3B is that the repressed maternal nos-2 mRNA is located in the embryonic P-granules, while FBF-2 can efficiently maintain the repressed state of its target mRNAs outside P-granules, provided the FBF-2/mRNA complex had a chance to form. These differences are likely to result from varying mechanisms of action and specific properties of the individual RNA regulatory proteins found in each repressive complex.
An exciting potential for P-granule function is offered by a recently discovered self/non-self recognition mechanism leading to preferential silencing of non-self transcripts triggered at the mRNA level in the C. elegans germline (Lee et al., 2012). Molecular players involved in self/non-self determination are the Argonautes PRG-1 and CSR-1, which are enriched in P-granules and hypothesized to function by recognizing the non-self transcripts (PRG-1) or anti-silencing the endogenous transcripts (CSR-1) based on transcripts' sequence complementarity to the Argonaute-associated small RNAs. Their localization in P-granules positions PRG-1 and CSR-1 ideally to screen the germ cell's transcripts for self/non-self RNA molecules upon their nuclear export. It would be interesting to determine if P-granules play any structural role in supporting the efficiency of this invader defense mechanism.
A NEW AVENUE TO EXPLORE: NUCLEO-CYTOPLASMIC TRANSPORT
The position of P-granules at the nuclear periphery overlapping the nuclear pores has led to a hypothesis that these organelles may be involved in the regulation of nucleo-cytoplasmic exchange in germ cells, in a way that may require specific mechanisms for mRNA transit (Schisa et al., 2001; Sheth et al., 2010; Updike et al., 2011). Interestingly, while there is no evidence for mRNA export or trafficking changes in P-granule mutants, a possibility has emerged that P-granules prevent nuclear re-import of a retrotransposon into the germ cell nuclei (Fig. 3C), which is likely mediated by a different mechanism than RNA regulation described earlier (Fig. 3A,B). A recently discovered C. elegans transposable element, CER-1, is activated at low temperatures in germ cells (Dennis et al., 2012). CER-1 forms virus-like capsids in the cytoplasm, which track back to germline nuclei, but then fail to enter and to activate transposition. CER-1 transposon capsids only accumulate near those germ cell nuclei that have available P-granule-free nuclear pores (Dennis et al., 2012). This raises an exciting possibility that P-granules protect germ cell nuclei from retroelement attack. If true, this would expand the known strategies of transposon defense employed by the germ cells of C. elegans as well as other animals, which are currently thought to be based on PIWI/piRNA-function (Juliano et al., 2011). It would be interesting to explore transposon activity in P-granule mutants to better characterize this potential function.
CONCLUSIONS AND OPEN QUESTIONS
Current research on P-granules highlights many functions of these organelles, perhaps accounting for the variety of developmental requirements for their presence. For the road ahead, characterizing the complete repertoire of P-granule components, including proteins and RNAs, would help to better understand the various processes they govern. Beyond the function of individual components, an important goal is to explore the role of P-granule structure proper. A number of proteins accumulating in P-granules appear to gain functionality via this localization, and it would be instructive to determine if this is a general phenomenon and if each component derives a specific advantage from its localization to P-granules. Answering these questions will further inform us of the contribution of this spatially organized germ cell regulatory machinery to the development and function of germ cells.
The author would like to thank the reviewers for helpful comments and critiques and to apologize to those whose work was not discussed due to space constraints.
Research support is provided by the University of Montana startup funds.