Next Generation Organelles: Structure and Role of Germ Granules in Germline

SUMMARY

Germ cells belong to a unique class of stem cells which give rise to eggs and sperm and ultimately to an entire organism after the fusion of the gametes. In many organisms, germ cells contain electron-dense structures also known as nuage or germ granules. Although the germ granules were discovered more than 100 years ago, their composition, structure, assembly and function are not fully understood. Germ granules contain noncoding RNAs, mRNAs and proteins required for germline development. Here we review recent studies which highlighted the importance of several protein families in germ granule assembly and function, including germ granule inducers, which initiate the granule formation, and downstream components such as RNA helicases and Tudor domain - Piwi protein - piRNA complexes. Assembly of these RNAs and proteins in one granule is likely to result in a highly efficient molecular machine which ensures translational control and protects germline DNA from mutations caused by mobile genetic elements. Furthermore, recent studies have shown that different somatic cells, including stem cells and neurons, produce germ granule components which play a crucial role in stem cell maintenance and memory formation, indicating a much more diverse functional potential of the granules than previously thought.

HISTORICAL BACKGROUND

Early developmental studies suggested that the reproduction of organisms was achieved by the continuity of germ cells from one generation to the next, during which some of the germ cells took the path of differentiation to form the somatic cells while some other germ cells remain totipotent and contribute to the development of the progeny to come (Nussbaum, 1880; Wilson, 1896). Subsequent investigations found evidence suggesting that, rather than the continuity of the germ cells, it was the continuity of substances passed down from the parent germ cells to the germ cells of the progeny that ensured the heredity of the species, with this substances named ‘germ plasm’ (Weismann, 1892). The first evidence supporting this germ plasm theory came from the successful tracing of granules from the posterior pole cytoplasm of insect oocytes in one generation to the germ cells of the next generation, with the substances referred to as ‘germ cell determinants’ (Hegner, 1914). Also, the term ‘chromatoid body’ was introduced by early investigators to describe a germ cell organelle in mammalian spermatocytes and spermatids based on the fact that this structure exhibits similar staining characteristics as chromosomes and nucleoli when examined under light microscope (Benda, 1891; Hermann, 1889; Yokota, 2008). The employment of electron microscope techniques from the 1950s to 1970s led to a more detailed morphological description of the germ cell-specific structures: high electron density, granular or fibrous in shape, no confining membrane, frequently surrounded by small vesicles and usually accompanied by mitochondria or associated with nuclear envelope (al-Mukhtar and Webb, 1971; Brokelmann, 1963; Eddy, 1974; Fawcett et al., 1970; Mahowald, 1962; Russell and Frank, 1978). Therefore, the term ‘nuage’ (meaning cloud in French) was also used to describe this dense material (André and Rouiller, 1956). From the 1970s, much histochemical study on nuage has been done to investigate its composition at the molecular level. Despite early discrepancies on whether RNA is a component of nuage, solid evidence from RNase treatment experiments and in situ hybridization studies suggested that RNA, at some developmental stages of germ cells, is contained in nuage (Afzelius, 1957; Conway, 1971; Daoust and Clermont, 1955; Eddy, 1970; Eddy and Ito, 1971; Kotaja et al., 2006; Mahowald, 1971; Sud, 1961b). The presence of proteins in nuage was confirmed by trichloroacetic acid treatment experiments and labeled amino acid incorporation studies (Eddy and Ito, 1971; Sud, 1961a; Sud, 1961b). Later around 1975, a series of transplantation experiments were performed to demonstrate the function of germ plasm (posterior pole cytoplasm) from Drosophila. The transplanting of intact germ plasm successfully restored the germ cell formation ability of UV-irradiated embryos (Okada et al., 1974). Furthermore, the transplantation of the germ plasm from the posterior pole to the anterior pole and the midventral site in Drosophila eggs resulted in the formation of germ cells at these sites (Illmensee and Mahowald, 1974; Illmensee and Mahowald, 1976).

A variety of different names for germline-specific electron-dense structures have been given to describe their different morphological features, intracellular localization and organism origin. This variation may indicate that different germ cell granules play some unique roles besides their similar functions in germline development. In particular, in addition to nuage and chromatoid body, the following structures have been described: P granules in Caenorhabditis elegans, polar granules in the germ plasm of Drosophila, intermitochondrial cement in Xenopus and mouse oocytes and mouse spermatocytes (Bilinski et al., 2004; Chuma et al., 2009; Eddy, 1975; Kloc et al., 2004; Seydoux and Braun, 2006). In this article, based on the structural, biochemical and functional similarities of the germ plasm-like structures mentioned above, we will generally use the term ‘germ granules’ when referring to them unless otherwise specified.

COMPOSITION AND FUNCTION OF GERM GRANULES

Germ granules in different organisms are composed of both RNA and protein components (Arkov and Ramos, 2010; Schisa, 2012; Voronina et al., 2011) (Table 1) and identification and characterization of these components has pointed to some of the presumed functional roles of the granules in germline development including translational control and protection of germline DNA from mutations caused by transposon insertions. Interestingly, it has been shown that in C. elegans, formation of maternal germ granules during embryogenesis is not essential for germline specification but it is required for complete fertility at high temperatures (Gallo et al., 2010). Therefore, in C. elegans, the assembly of maternal germ granules in developing germ cells may serve as a protection mechanism against stress.

Table 1.

Prominent Germ Granule/Nuage Components

Proteins (function)	Organism	Germ Granule Type	References
RNA helicases (RNA unwinding; translational control):
Vasa	Fruit fly	perinuclear nuage and polar granules	(Hay et al., 1988; Lasko and Ashburner, 1990)
Belle	Fruit fly	perinuclear nuage	(Johnstone et al., 2005)
Me31B	Fruit fly	polar granules	(Thomson et al., 2008)
eIF4A	Fruit fly	polar granules	(Thomson et al., 2008)
Armitage	Fruit fly	perinuclear nuage	(Cook et al., 2004)
GLH-1-4 proteins	C. elegans	P granules	(Kuznicki et al., 2000)
CGH-1	C. elegans	P granules	(Navarro et al., 2001)
XVLG1	Frog	perinuclear nuage intermitochondrial cement	(Bilinski et al., 2004)
Vasa	Zebrafish	primordial germ cell perinuclear granules	(Knaut et al., 2000)
Mvh	Mouse	chromatoid body	(Toyooka et al., 2000)
DDX25/GRTH	Rat	chromatoid body	(Onohara and Yokota, 2012)
Tudor Domain-containing proteins (scaffold function; transposon silencing):
Tudor	Fruit fly	perinuclear nuage and polar granules	(Bardsley et al., 1993)
SpnE	Fruit fly	perinuclear nuage	(Anand and Kai, 2012)
Krimp/Mtc	Fruit fly	perinuclear nuage	(Barbosa et al., 2007; Lim and Kai, 2007)
Tejas	Fruit fly	perinuclear nuage	(Patil and Kai, 2010)
PAPI	Fruit fly	perinuclear nuage	(Liu et al., 2011)
Qin/Kumo	Fruit fly	perinuclear nuage	(Anand and Kai, 2012; Zhang et al., 2011)
Brother of Yb	Fruit fly	perinuclear nuage	(Handler et al., 2011)
Tdrd1/Mtr-1, Tdrd5, Tdrd6, Tdrd7	Mouse	intermitochondrial cement chromatoid body	(Chuma et al., 2009; Tanaka et al., 2011; Vagin et al., 2009; Yabuta et al., 2011)
Tdrd9	Mouse	chromatoid body piP-bodies	(Aravin et al., 2009; Shoji et al., 2009)
Tdrdl, Tdrd7	Zebrafish	primordial germ cell granules	(Huang et al., 2011; Strasser et al., 2008)
Piwi family proteins (transposon silencing; translational control):
Aubergine	Fruit fly	perinuclear nuage and polar granules	(Harris and Macdonald, 2001; Thomson et al., 2008)
Argonaute 3 (Ago3)	Fruit fly	perinuclear nuage	(Brennecke et al., 2007; Gunawardane et al., 2007)
PRG-1	C. elegans	P granules	(Batista et al., 2008; Wang and Reinke, 2008)
Miwi	Mouse	chromatoid body	(Vagin et al., 2009)
Mili	Mouse	intermitochondrial cement chromatoid body	(Aravin et al., 2009; Vagin et al., 2009)
Miwi2	Mouse	piP-bodies	(Aravin et al., 2009)
Ziwi	Zebrafish	primordial germ cell granules	(Houwing et al., 2007)
Maelstrom proteins (transposon silencing)	Fruit fly	perinuclear nuage	(Findley et al., 2003; Lim and Kai, 2007)
	Mouse	chromatoid body	(Costa et al., 2006; Soper et al., 2008)
Sm proteins (spliceosome components)	Fruit fly	polar granules	(Anne, 2010; Gonsalvez et al., 2010)
	C. elegans	P granules	(Barbee et al., 2002)
	Mouse	chromatoid body	(Chuma et al., 2003)
Germ granule inducers (initiation of germ granule formation by recruiting downstream granule components):
Oskar	Fruit fly	polar granules	(Vanzo et al., 2007)
PGL-1, PGL-3	C. elegans	P granules	(Hanazawa et al., 2011; Updike et al., 2011)
Bucky Ball	Zebrafish	germ plasm granules	(Bontems et al., 2009)
RNAs (function)
mRNAs (coding for germline proteins): nanos	Fruit fly	polar granules	(Rangan et al., 2009)
Xcat2	Frog	germinal granules intermitochondrial cement	(Bilinski et al., 2004; Kloc et al., 2002; Mosquera et al., 1993)
nos-2	C. elegans	P granules	(Subramaniam and Seydoux, 1999)
gld-1	C. elegans	P granules	(Schisa et al., 2001)
mex-1	C. elegans	P granules	(Schisa et al., 2001)
pos-1	C. elegans	P granules	(Schisa et al., 2001)
polar granule component	Fruit fly	polar granules	(Hanyu-Nakamura et al., 2008; Nakamura et al., 1996)
germ cell-less	Fruit fly	polar granules	(Amikura et al., 2005)
Noncoding RNAs (translation):
mitochondrial large and	Fruit fly	polar granules	(Amikura et al., 2001)
small ribosomal RNAs	Frog	germinal granules	(Kashikawa et al., 2001)

Three classes of protein components are consistently found in germ granules - RNA helicases, Tudor (Tud) domain-containing proteins and Piwi family proteins (Table 1). In addition, many germline mRNAs as well as noncoding RNAs, for example, small Piwi-interacting RNAs (piRNAs) and mitochondrial ribosomal RNAs are present in the granules (Table 1). Furthermore, in several types of germ granules in fly, C. elegans and zebrafish, specific proteins have been identified which start the assembly of the granules by recruiting conserved granule components, such as Vasa (Vas) or its homologs. We refer to these proteins as ‘germ granule inducers’ (Table 1). It is remarkable that the germ granule inducers have not been evolutionarily conserved based on their amino acid sequences and are specific to certain insects (Oskar), worms (PGL proteins) or vertebrates (Bucky Ball) (Table 1). It will be interesting to determine if germ granule inducers can be swapped between different organisms and whether their tertiary structures are similar.

RNA Helicases

In many animals germ granules are very dynamic and they change during development. Since the granules are RNA-protein structures, it is likely that the dynamic changes are caused by the remodeling of these ribonucleoprotein (RNP) complexes. The active remodeling of RNP structures is consistent with the presence of multiple RNA helicases in germ granules (Table 1).

Germ granule RNA helicases bind to ATP and unwind double-stranded RNAs in the RNP particles, allowing them to acquire an alternative conformation (Linder and Lasko, 2006; Sengoku et al., 2006). Therefore, the helicases may be responsible for reshaping the granule architecture.

The functional role of RNA helicases in the germ granules has been well documented by the genetic analysis of mutations in the genes encoding the helicases in different organisms (Arkov and Ramos, 2010). In particular, Vas helicase and its homologs are crucial for germline development in many organisms (Gustafson and Wessel, 2010; Raz, 2000). In the fly, Vas is required for oogenesis and germ cell formation in the embryos (Linder and Lasko, 2006) and the mouse Vas homolog, Mvh, is necessary for spermatogenesis (Tanaka et al., 2000).

Tudor Domain Proteins

Many proteins containing Tud domains have been identified in germ granules from different model organisms (Table 1). Tud domain is a small 50-55 amino acid β-barrel module which forms a pocket lined with aromatic amino acids (aromatic cage) (Chen et al., 2011; Pek et al., 2012). The aromatic cage interacts with methylated amino acids, lysines or arginines of target proteins. Many germline Tud domain-containing proteins have extended Tud domains (eTud) which consist of the Tud core β-barrel with flanking α helices and β strands that form a staphylococcal nuclease (SN)-like fold (Chen et al., 2011; Liu et al., 2010). In addition, many germ granule Tud domain-containing proteins have multiple eTud domains (for example, fly Tud protein has 11 domains) (Arkov et al., 2006; Chen et al., 2011; Creed et al., 2010). Interestingly, in the same protein, Tud domain can be found with other domains which interact with RNA, including RNA helicase domains (in fly SpnE and in the mouse Tdrd9 protein) (Shoji et al., 2009).

In fly, mouse and zebrafish, Tud domain proteins have been shown to play crucial roles in germ granule assembly (Boswell and Mahowald, 1985; Chuma et al., 2006; Strasser et al., 2008; Vasileva et al., 2009). Also, fly Tud protein is essential for primordial germ cell formation (Arkov et al., 2006; Boswell and Mahowald, 1985; Thomson and Lasko, 2004) and the mouse Tud domain proteins function in spermatogenesis (Chuma et al., 2006; Pan et al., 2005; Vasileva et al., 2009).

Piwi Family Proteins

In germ granules, Tud domain-containing proteins interact with symmetrically dimethylated arginines (sDMAs) of Piwi family proteins in fly and mammals and these Tud - Piwi protein complexes safeguard germline genomes against retrotransposons (Creed et al., 2010; Kirino et al., 2010; Nishida et al., 2009; Siomi et al., 2010; Vagin et al., 2009; Vourekas et al., 2010). More specifically, in fly and mammals, antisense piRNAs bound to Piwi proteins guide these proteins to transposon mRNAs which are then cleaved by Piwi proteins (reviewed in (Juliano et al., 2011; Pek et al., 2012; Thomson and Lin, 2009)). However, a recent study in C. elegans uncovered Piwi endonuclease-independent mechanism of piRNA-mediated silencing, which depends on piRNA-induced secondary endogenous small interfering RNA (endo-siRNA) response (Bagijn et al., 2012). Therefore, while the molecular mechanism of transposon silencing may differ in different species, the requirement to protect germline DNA using piRNAs has been evolutionarily conserved.

In many organisms as diverse as fly, mouse, zebrafish, and C. elegans, Piwi proteins are required for fertility. In addition, in planarian flatworms and colonial ascidians, Piwi proteins are expressed in stem cells and required for regeneration (Juliano et al., 2011; Thomson and Lin, 2009).

RNA Components of Germ Granules

Germ granules have been suggested to control translation of a variety of germline mRNAs associated with the granules, including nanos (nos), polar granule component (pgc), and germ cell-less (gcl) (Mahowald, 1968; Rangan et al., 2009). Translational repression and activation of specific mRNAs have been proposed to occur in these granules at distinct time points during development since differentially translated mRNAs do not dissociate from the granules irrespective of their translational status (Rangan et al., 2009). Furthermore, the mRNA 3′-untranslated regions (3′UTRs) have an instructive role for both mRNA localization to germ granules and the translational status of these mRNAs. It is likely that the translation activation or repression outcome is the result of highly coordinated interplay between 3′UTRs and specific RNA-binding protein factors, including RNA helicases, in germ granules.

In addition to mRNAs, germ granules contain noncoding RNAs (Table 1). In particular, piRNAs are expected to localize to the granules since these RNAs directly associate with granule Piwi family proteins. Also, RNAs from the large and small subunits of mitochondrial ribosomes are germ granule components (Table 1). In fly, there is evidence that mitochondria-type ribosomes are involved in translation of gcl mRNA on polar granules (Amikura et al., 2005). These intriguing data suggest an exciting possibility for the important role of mitochondrial translation in the cytosol during primordial germ cell formation.

ASSEMBLY AND SHAPING OF THE GERM GRANULES

The detailed pathway of germ granule assembly has not been fully deciphered. Therefore, here we will review some of the most recent data obtained from various model organisms. These results will lead to a better understanding of the factors which determine the fate of germ granules and help to maintain their integrity.

Germ granule assembly in Drosophila oocyte’s germ plasm

Drosophila germ granule assembly in posterior germ plasm occurs through a series of hierarchical events that center at the transport of oskar (osk) mRNA to the posterior pole of the oocyte (Ephrussi et al., 1991; Kim-Ha et al., 1991). The correct localization of osk mRNA relies on the organization and the reorganization of microtubules in the egg chamber during early oogenesis (Steinhauer and Kalderon, 2006). In stage 2 - 6 oocyte, the microtubules expand their plus-ends via the ring canals into the nurse cells and group their minus-ends in the oocyte, with the microtubule organizing center (MTOC) positioned to the posterior cortex of the oocyte, directing the transport of maternal mRNAs (for example, grk mRNA) synthesized by nurse cells to the posterior pole of the transcriptionally inactive oocyte (Grieder et al., 2000; Saunders and Cohen, 1999; Theurkauf et al., 1992). After stage 6, the germline microtubules undergo rearrangement triggered by the signaling between the oocyte and the follicle cells adjacent to the posterior pole of the oocyte. This rearrangement results in the disassembly of the posterior MTOC and the subsequent appearance of microtubules from the entire oocyte cortex, pointing their plus-ends towards the center of the oocyte (Cha et al., 2001; Cha et al., 2002; Steinhauer and Kalderon, 2006). At stage 8 - 9, the posterior cortical microtubules are disassembled, followed by the microtubule plus-ends reorientation towards the posterior pole (Cha et al., 2002; Dollar et al., 2002). Consequently, osk mRNA begins to accumulate at the posterior pole via the Kinesin I-dependent transport, dictating the location of the germ plasm formation (Brendza et al., 2000; Ephrussi and Lehmann, 1992). Interestingly, instead of moving in ‘one-way traffic’, osk mRNA exhibits a ‘random walk’ in all directions with a weak bias towards the posterior pole (Zimyanin et al., 2008). The enrichment of microtubule plus-ends has been suggested to lead to the translational derepression of osk mRNA (Becalska and Gavis, 2010). The resulted Osk protein then initiates germ plasm assembly by recruiting other germ plasm components (for example, Vas and Tud) to the posterior pole. In particular, the direct interaction between Osk and Vas has been experimentally established and recognized as an initial step in germ plasm assembly (Breitwieser et al., 1996).

In addition to flies and other dipterans, a recent study has identified osk gene in some other insect species which exhibit maternal germ plasm assembly and concluded that the evolutionary origin of osk in insects has been correlated with the appearance of the germ plasm (Lynch et al., 2011).

Although the mechanisms governing the reorganization of microtubule cytoskeleton during mid-oogenesis remain unclear, recent research on Bazooka (Baz), the Drosophila homolog of Par-3 (abnormal embryonic PARtitioning of cytoplasm-3), revealed a novel role for Baz in regulating oocyte microtubule polarity (Becalska and Gavis, 2010). Baz localizes to the anterior and lateral cortex and is excluded from the posterior pole (Becalska and Gavis, 2010; Benton and St Johnston, 2003). Therefore, it is suggested that Baz might exert its influence on restricting Osk-induced germ plasm assembly at the posterior pole by controlling microtubule polarity to ensure microtubule plus-ends reaching to posterior (Becalska and Gavis, 2010) (Fig. 1).

Figure 1.

Illustration of microtubule activity, the localization of osk mRNA and Baz protein during initiation of germ plasm assembly in Drosophila oocyte. During oogenesis stage 2 to 6, microtubules group the minus-ends in the oocyte and reach the plus-ends into nurse cells. In stage 6 to 7, microtubules appear from the entire oocyte cortex and rearrange so that the plus-ends cluster towards the center of the oocyte, along with the disassembly of early-stage microtubule organizing center (MTOC). At stage 8 to 9, microtubules at the posterior pole disassemble, followed by the relocation of microtubule plus-ends to the posterior pole. This microtubule reorganization during mid-oogenesis requires the functionality of Baz, which localizes to the anterior and lateral cortex. At stage 9, osk mRNA is transported to the posterior pole and its translation derepression is initiated by the accumulation of microtubule plus-ends. Then, the resulted Osk protein starts the germ plasm assembly by recruiting other germ plasm components such as Vas and Tud (Becalska and Gavis, 2010; Breitwieser et al., 1996; Ephrussi and Lehmann, 1992; Steinhauer and Kalderon, 2006). (Part of the figure is modified from Steinhauer and Kalderon, 2006).

Germ Plasm Assembly in Zebrafish

The germ plasm in oocytes of vertebrate animals is found in a cellular structure called the Balbiani body (Heasman et al., 1984; Kloc et al., 2004; Mahowald, 2001; Marlow and Mullins, 2008; Pepling et al., 2007; Strome and Lehmann, 2007). Although the assembly machinery of the Balbiani body germ plasm has not been fully elucidated, recent research on the Balbiani body in zebrafish identified bucky ball (buc) gene as a key germ plasm inducer that governs the germ plasm assembly in the Balbiani body (Bontems et al., 2009; Marlow and Mullins, 2008). The buc mutant was identified by its defect in germ plasm localization and polarity (Dosch et al., 2004; Marlow and Mullins, 2008). Furthermore, key germ plasm markers dazl (Maegawa et al., 1999), vas (Olsen et al., 1997), and nos mRNA (Koprunner et al., 2001) fail to localize to the Balbiani body in the buc mutant (Bontems et al., 2009). Despite very limited functional domain information from bioinformatics approach, Buc protein has been shown to initially localize to the Balbiani body then gradually to the vegetal pole in the oocyte, where it is proposed to recruit the downstream germ plasm components including dazl, vas, and nos mRNA (Bontems et al., 2009) (Fig. 2). The competence of Buc to form germ plasm is further supported by the induction of additional but ectopic germ cells upon Buc overexpression in the embryo, with the hypothesized mechanism by which Buc aggregates germ plasm components already present in the early embryo and prevents their degradation during embryogenesis (Bontems et al., 2009).

Figure 2.

Buc protein activity during germ plasm assembly in zebrafish oocyte. Buc (indicated in red) localizes to the Balbiani body in the oocyte and functions to recruit germ plasm RNA components including dazl, vas, and nos. During development, Buc gradually spreads within the vegetal pole (Bontems et al., 2009).

P granules assembly in C. elegans

Similar to fly and zebrafish, germ granule assembly in C. elegans is directed by worm-specific PGL (P-GranuLe abnormality) proteins which are responsible for the recruitment of the downstream germ plasm components (Updike et al., 2011). Recent research revealed critical roles of PGL proteins and GLH (Germ Line Helicase) proteins (Vas homologs) in the assembly of the granules. In the absence of other germline-specific factors, PGL-1 (Kawasaki et al., 1998) and PGL-3 (Kawasaki et al., 2004) proteins are able to self-associate to form germ granule-like aggregates. The aggregation requires the N-terminal self-interacting domains (Hanazawa et al., 2011). Also, the PGL proteins’ C-terminal RGG boxes act as RNA-binding domains that incorporate RNAs and RNPs into the germ granules (Godin and Varani, 2007; Hanazawa et al., 2011) (Fig. 3).

Figure 3.

PGL proteins utilize the two types of functional domains to start formation of P granule-like aggregates. In step 1, RNAs and RNA binding proteins exist as small RNPs. In step 2, PGL proteins use the RGG box to bind to the RNA components in the RNPs. In step 3, PGL proteins associated with RNPs begin self-aggregation through the self-interacting domains. In step 4, PGL protein self-aggregation continues to build P granules (Hanazawa et al., 2011).

GLH-1, GLH-2, and GLH-4 proteins in worm, contain N-terminal phenylalanine-glycine (FG) repeat domains, which are likely to interact to establish a hydrophobic network of filaments, mimicking the role of FG repeats in nuclear pore complex (NPC) proteins (Patel et al., 2007; Ribbeck and Gorlich, 2002; Updike et al., 2011). Work done on GLH proteins uncovered that GLH-1 and GLH-3 cannot form granules by themselves unless additional FG domains are inserted or PGL-1 is present. These results support the idea that granules can form only after reaching a local FG-repeat concentration threshold, which in turn is provided by the nucleating effect of self-aggregating PGL-1 (Hanazawa et al., 2011; Pappu et al., 2008; Ribbeck and Gorlich, 2002; Updike et al., 2011). Furthermore, GLH-1 has been suggested to retain the perinuclear localization of PGL proteins and help to lower the saturation point of free PGL proteins in the germline thereby favoring the subsequent granule assembly. These data demonstrate the interplay between GLH and PGL proteins in maintaining the granule structure (Brangwynne et al., 2009; Hanazawa et al., 2011; Updike et al., 2011).

A recent research on the physical nature of P granules in C. elegans provided significant insight on the shaping of germ granules. It was shown that the P granules behave as liquid droplets, bearing a viscosity roughly 1000 times that of water (similar to the viscosity of glycerol) and a very small surface tension value which is typical for macromolecular liquids. These liquid droplet-like physical properties enable asymmetric cytoplasmic distribution of P granules by dissolution from anterior and condensation towards posterior upon the symmetry breaking in C. elegans embryos (Brangwynne et al., 2009).

Role of Tudor and Piwi Proteins in Germ Granule Assembly

The presence of scaffold factors in the assembly of germ granules has been reported in different organisms, for example, Tud domain-containing proteins in fly, zebrafish and mouse (Arkov et al., 2006; Huang et al., 2011; Vasileva et al., 2009). As outlined above, Tud domain-containing proteins frequently have multiple Tud domains (Chen et al., 2011). In particular, fly Tud protein plays an important role in the germ granules by binding to sDMAs of Piwi protein Aubergine (Aub) (Kirino et al., 2010; Nishida et al., 2009). The cocrystallization of heterologously expressed Tud domain 11 of Tud protein and sDMA-containing Aub peptide revealed the recognition nature of sDMA ligand by an asparagine-gated aromatic cage (Liu et al., 2010). Considering the fact that multiple Tud domains contribute to the binding between Tud and Aub (Creed et al., 2010), and that Piwi proteins tend to contain multiple sDMA motifs (Kirino et al., 2009; Kirino et al., 2010), it has been proposed that Tud-domain proteins have a scaffold function whereby the Tud domains serve as docking platforms for the assembly of macromolecules during germ granule formation (Arkov et al., 2006; Chen et al., 2011) (Fig. 4).

Figure 4.

Germ granule assembly through Tudor-Piwi interaction. The Tudor-Piwi interactions may exhibit a combination of several possible binding patterns as follows. First, a Piwi protein contains one sDMA which binds to one Tud domain. Second, a Piwi protein contains multiple sDMAs each of which binds to one Tud domain on the same Tud domain-containing protein. Third, a Piwi protein contains multiple sDMAs each of which binds to one Tud domain on different Tud domain-containing proteins (Chen et al., 2011).

GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS

Germ granule components are not restricted to the germline. The most studied non-germline nuage-like structures are the chromatoid bodies in planarian neoblasts, a special population of somatic stem cells responsible for the animal’s regeneration and homeostasis (Newmark and Sanchez Alvarado, 2000; Newmark and Sanchez Alvarado, 2002). Several conserved germ granule components have been confirmed in the neoblasts’ chromatoid bodies, namely CBC-1, the DEAD box RNA helicase (Yoshida-Kashikawa et al., 2007); SmB, the small nuclear ribonucleoprotein (snRNP) core protein (Fernandez-Taboada et al., 2010); Spoltud, the Tud homolog (Solana et al., 2009). Also, SMEDWI-3, an sDMA-containing Piwi protein, is very likely another chromatoid body component (Rouhana et al., 2012). In addition, other Piwi proteins including SMEDWI-1 and SMEDWI-2, piRNA-like RNA populations and protein arginine methyltransferase 5 (PRMT5) are also found in neoblasts (Palakodeti et al., 2008; Reddien et al., 2005; Rouhana et al., 2012), reminiscent of the Piwi-piRNA-Tud germ granule assembly mechanism discussed above (Rouhana et al., 2012).

Notably, there is rising focus on the novel role of Piwi-piRNA complexes in non-germline cells, especially neurons. In mice, for example, a particular piRNA and Miwi protein have been shown to colocalize to the hippocampal neurons, controlling the dendritic spine morphogenesis (Lee et al., 2011). Furthermore, in the neurons of Aplysia, specific piRNAs associate with Piwi, and the piRNA-Piwi complex downregulates expression of CREB2, which is a memory repressor (Rajasethupathy et al., 2012). Tdrd7, a Tud domain-containing protein component of the chromatoid body in mouse testis, is also expressed in lens fiber cells and tdrd7 loss-of-function mutation results in cataract as well as glaucoma (Lachke et al., 2011).

The assembly and maintenance mechanism of the germ granule-like RNPs in somatic cells is intriguing. A very recent work on cell-free formation of RNP granules provided convincing evidence supporting a prion-like mechanism of their assembly. In particular, it was shown that a specific chemical, isoxazole, when added to cell lysates of different origins, is able to trigger the formation of RNP precipitates highly enriched in RNA-binding proteins and associated mRNAs commonly found in other RNA granules. Furthermore, the protein constituents in the precipitates contained Low Complexity (LC) sequences and RNA-binding domains responsible for the reversible cell-free formation of amyloid-like fiber aggregates and the recognition of the 3′-UTRs of their target mRNAs respectively. Therefore, it has been hypothesized that the cell-free RNPs employ a prion-like assembly mechanism which might also be the general principle guiding the assembly of other similar subcellular, non-membrane bound RNA granules (Han et al., 2012; Kato et al., 2012) (Fig. 5). An intensely studied example of mRNA binding protein which exhibits prion-like properties is the neuronal isoform of Aplysia cytoplasmic polyadenylation element binding protein (CPEB). Earlier studies on Aplysia CPEB in yeast cells demonstrated that its N-terminal prion-like domain (PrD) contributes to self-multimerization ability, self-sustainability and transmissibility, all of which are canonical characteristics of prion-like proteins (Alberti et al., 2009; Si et al., 2003). Contrary to the prion-like RNP assembly paradigm discussed above, the aggregated conformational state of CPEB, rather than its monomeric state, binds to its target mRNA (Si et al., 2003). Subsequent studies of Aplysia CPEB in neuron cells and its Drosophila homolog Orb2 further showed that multimerization of CPEB/Orb2 in the nerve system is regulated by specific stimuli, confirming their conserved roles in long-term synaptic facilitation (Si et al., 2010). Another phenomenon supporting the prion-like RNP assembly mechanism is that a cytoplasmic population of the Prion Protein (cyPrP) expressed in neurons induces the formation of an RNA organelle (PrP-RNP) which shares significant similarities with chromatoid bodies from the germline, including their RNA and protein components, clustering with mitochondria, dependence on microtubule network for assembly, and proximity to nuclear pore complexes (Beaudoin et al., 2009).

Figure 5.

Prion-like germ granule assembly mechanisms exemplified by RNA binding proteins containing Low Complexity (LC) sequences and RNA binding domains. The LC sequences enable the RNA binding proteins to exist in one of the following three states: a monomeric, soluble state; a polymeric, amyloid-like fiber state; or a pathogenic aggregate state. The transition between the first two states is reversible, allowing RNA binding proteins to enter or exit the prion-like aggregated cellular structure. However, the conversion to the third state is irreversible and pathogenic (Han et al., 2012; Kato et al., 2012).

CONCLUSIONS AND FUTURE OUTLOOK

Germ granules from different organisms are highly dynamic and complex RNP structures. While multiple granule components have been discovered, the complete granule composition is still far from being understood. Future research should result in a more comprehensive list of germ granule components and importantly how the composition of the granules changes during development in different organisms, and how these changes contribute to germline development.

Several germ granule inducers, which initiate the assembly of some types of granules, have been discovered, including Osk, PGL proteins, and Buc (Table 1). It is remarkable that even though these proteins recruit similar downstream granule components (for example, Vas or Vas homologs), they are different if their sequences are compared, and specific to particular groups of organisms. Structural analysis of germ granule inducers and their interacting partners will shed light on the early steps of the granule assembly and may reveal exciting structural similarities in different granule inducers.

Recent studies have highlighted the consistent presence of RNA helicases, Tud domain-containing proteins and Piwi - piRNA complexes in germ granules from different organisms and the role of Tud - Piwi - piRNA complexes in protection of germline DNA from transposons (Table 1). In addition, details of molecular recognition of Piwi proteins’ symmetrically dimethylated arginines (sDMAs) by Tud domains in germ granules have been a focus of intense and exciting research. Since germ granules frequently contain proteins with multiple Tud domains which interact with Piwi proteins, it will be important to determine the stoichiometry and structure of such a Tud - Piwi complex. The structural analysis should provide insights into the architecture of this crucial component of germ granules and may explain the functional significance of Tud - Piwi interaction.

Recent discoveries of germ granule components in somatic cells, including stem cells and neurons, are exciting indications that these granule components might assemble in different cells and these assemblies play important roles in stem cell maintenance and memory formation mechanisms. It will be interesting to determine if granule components in different cell contexts follow the same rules of assembly and to explore the functional significance of building a germ-like granule in somatic cells.

Abbreviations

RNP

ribonucleoprotein

sDMA

symmetrically dimethylated arginine

3′UTR

3′-untranslated region

MTOC

microtubule organizing center

NPC

nuclear pore complex