SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Germ cells belong to a unique class of stem cells that gives rise to eggs and sperm, and ultimately to an entire organism after gamete fusion. In many organisms, germ cells contain electron-dense structures that are also known as nuage or germ granules. Although germ granules were discovered more than 100 years ago, their composition, structure, assembly, and function are not fully understood. Germ granules contain non-coding RNAs, mRNAs, and proteins required for germline development. Here we review recent studies that highlight 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 components into one granule is likely to result in a highly efficient molecular machine that 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 that play a crucial role in stem cell maintenance and memory formation, indicating a much more diverse functional repertoire for these organelles than previously thought. Mol. Reprod. Dev. 80:610–623, 2013. © 2012 Wiley Periodicals, Inc.


Abbreviations
3′-UTR

3′-untranslated region

RNP

ribonucleoprotein

sDMA

symmetrically dimethylated arginine

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES

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 future progeny (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's germ cells to the germ cells of the progeny that ensured the heredity of the species, hence the collective term “germ plasm” to represent these substances (Weismann, 1892).

The first evidence supporting a germ plasm model 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 exhibits structure similar to chromosomes and nucleoli when examined under a 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 the nuclear envelope (Mahowald, 1962; Brokelmann, 1963; Fawcett et al., 1970; al-Mukhtar and Webb, 1971; Eddy, 1974; 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 of nuage has been done to investigate its composition at the molecular level. Despite early discrepancies as to whether or not 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 (Daoust and Clermont, 1955; Afzelius, 1957; Sud, 1961b; Eddy, 1970; Conway, 1971; Eddy and Ito, 1971; Mahowald, 1971; Kotaja et al., 2006). The presence of proteins in nuage was confirmed by trichloroacetic acid treatment experiments and labeled amino acid incorporation studies (Sud, 1961a, 1961b; Eddy and Ito, 1971). 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 ability of UV-irradiated embryos to form germ cells (Okada et al., 1974). Furthermore, transplantation of 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, 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 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, and intermitochondrial cement in Xenopus and mouse germ cells (Eddy, 1975; Bilinski et al., 2004; Kloc et al., 2004; Seydoux and Braun, 2006; Chuma et al., 2009). 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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Germ granules in different organisms are composed of both RNA and protein elements (Arkov and Ramos, 2010; Voronina et al., 2011; Schisa, 2012) (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 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
 OrganismGerm granule typeRefs.
Proteins (function)
RNA helicases (RNA unwinding; translational control)
VasaFruit flyPerinuclear nuage Polar granulesHay et al. (1988) and Lasko and Ashburner (1990)
BelleFruit flyPerinuclear nuageJohnstone et al. (2005)
Me31BFruit flyPolar granulesThomson et al. (2008)
eIF4AFruit flyPolar granulesThomson et al. (2008)
ArmitageFruit flyPerinuclear nuageCook et al. (2004)
GLH-1-4 proteinsC. elegansP granulesKuznicki et al. (2000)
CGH-1C. elegansP granulesNavarro et al. (2001)
XVLG1FrogPerinuclear nuage Intermitochondrial cementBilinski et al. (2004)
VasaZebrafishPrimordial germ cell perinuclear granulesKnaut et al. (2000)
MvhMouseChromatoid bodyToyooka et al. (2000)
DDX25/GRTHRatChromatoid bodyOnohara and Yokota (2012)
Tudor domain-containing proteins (scaffold function; transposon silencing)
TudorFruit flyPerinuclear nuage Polar granulesBardsley et al. (1993)
SpnEFruit flyPerinuclear nuageAnand and Kai (2012)
Krimp/MtcFruit flyPerinuclear nuageBarbosa et al. (2007) and Lim and Kai (2007)
TejasFruit flyPerinuclear nuagePatil and Kai (2010)
PAPIFruit flyPerinuclear nuageLiu et al. (2011)
Qin/KumoFruit flyPerinuclear nuageAnand and Kai (2012) and Zhang et al. (2011)
Brother of YbFruit flyPerinuclear nuageHandler et al. (2011)
Tdrd1/Mtr-1, Tdrd5, Tdrd6, Tdrd7MouseIntermitochondrial cement Chromatoid bodyChuma et al. (2009), Tanaka et al. (2011), Vagin et al. (2009) and Yabuta et al. (2011)
Tdrd9MouseChromatoid body, piP-bodiesAravin et al. (2009) and Shoji et al. (2009)
Tdrd1, Tdrd7ZebrafishPrimordial germ cell granulesHuang et al. (2011) and Strasser et al. (2008)
Piwi family proteins (transposon silencing; translational control)
AubergineFruit flyPerinuclear nuage Polar granulesHarris and Macdonald (2001) and Thomson et al. (2008)
Argonaute 3 (Ago3)Fruit flyPerinuclear nuageBrennecke et al. (2007) and Gunawardane et al. (2007)
PRG-1C. elegansP granulesBatista et al. (2008) and Wang and Reinke (2008)
MiwiMouseChromatoid bodyVagin et al. (2009)
MiliMouseIntermitochondrial cement Chromatoid bodyAravin et al. (2009) and Vagin et al. (2009)
Miwi2MousepiP-bodiesAravin et al. (2009)
ZiwiZebrafishPrimordial germ cell granulesHouwing et al. (2007)
Maelstrom proteins (transposon silencing)Fruit flyPerinuclear nuageFindley et al. (2003) and Lim and Kai (2007)
 MouseChromatoid bodyCosta et al. (2006) and Soper et al. (2008)
Sm proteins (spliceosome components)Fruit flyPolar granulesAnne (2010) and Gonsalvez et al. (2010)
 C. elegansP granulesBarbee et al. (2002)
 MouseChromatoid bodyChuma et al. (2003)
Germ granule inducers (initiation of germ granule formation by recruiting downstream granule components)
OskarFruit flyPolar granulesVanzo et al. (2007)
PGL-1, PGL-3C. elegansP granulesHanazawa et al. (2011) and Updike et al. (2011)
Bucky BallZebrafishGerm plasm granulesBontems et al. (2009)
RNAs (function)
mRNAs (coding for germline proteins)
nanosFruit flyPolar granulesRangan et al. (2009)
Xcat2FrogGerminal granules Intermitochondrial cementBilinski et al. (2004), Kloc et al. (2002), and Mosquera et al. (1993)
nos-2C. elegansP granulesSubramaniam and Seydoux (1999)
gld-1C. elegansP granulesSchisa et al. (2001)
mex-1C. elegansP granulesSchisa et al. (2001)
pos-1C. elegansP granulesSchisa et al. (2001)
polar granule componentFruit flyPolar granulesHanyu-Nakamura et al. (2008), Nakamura et al. (1996)
germ cell-lessFruit flyPolar granulesAmikura et al. (2005)
Noncoding RNAs (translation)
Mitochondrial large andFruit flyPolar granulesAmikura et al. (2001)
small ribosomal RNAsFrogGerminal granulesKashikawa 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 non-coding 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 that 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 appear to cluster by organism—for example, there are inducers that are specific to 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 or if 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 dynamic changes are related to 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 granule architecture.

The functional role of RNA helicases in 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 (Raz, 2000; Gustafson and Wessel, 2010). In the fly, Vas is required for oogenesis and germ cell formation in the embryos (Linder and Lasko, 2006) while the mouse Vas homolog, Mvh, is necessary for spermatogenesis (Tanaka et al., 2000).

Tudor Domain Proteins

Many proteins containing Tudor (Tud) domains have been identified in germ granules from different model organisms (Table 1). The Tud domain is a small, 50–55 amino acid β-barrel module that forms a pocket lined with aromatic amino acids (forming an aromatic cage) (Chen et al., 2011; Pek et al., 2012). The aromatic cage interacts with methylated amino acids, for example, 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 (Liu et al., 2010; Chen et al., 2011). In addition, many germ granule Tud domain-containing proteins have multiple eTud domains (e.g., fly Tud protein has 11 eTud domains) (Arkov et al., 2006; Creed et al., 2010; Chen et al., 2011). Interestingly, in the same protein, Tud domains can be found with other domains that interact with RNA, including RNA helicase domains (SpnE in fly and Tdrd9 in the mouse) (Shoji et al., 2009).

In the fly, mouse, and zebrafish, Tud domain proteins 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 (Boswell and Mahowald, 1985; Thomson and Lasko, 2004; Arkov et al., 2006) while the mouse Tud domain proteins function in spermatogenesis (Pan et al., 2005; Chuma et al., 2006; 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 the fly and mammals, and these Tud–Piwi protein complexes safeguard germline genomes against retrotransposons (Nishida et al., 2009; Vagin et al., 2009; Creed et al., 2010; Kirino et al., 2010; Siomi et al., 2010; Vourekas et al., 2010). More specifically in fly and mammals, antisense piRNAs bound to Piwi proteins guide these proteins to transposon mRNAs that are then cleaved by Piwi proteins (reviewed in Thomson and Lin, 2009; Juliano et al., 2011; Pek et al., 2012). A recent study in C. elegans, however, uncovered a Piwi endonuclease-independent mechanism of piRNA-mediated silencing that 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 organisms as diverse as fly, mouse, zebrafish, and C. elegans, Piwi proteins are required for fertility. In planarian flatworms and colonial ascidians, Piwi proteins are additionally expressed in stem cells and are required for regeneration (Thomson and Lin, 2009; Juliano et al., 2011).

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 translational activation or repression is the result of highly coordinated interplay between 3′-UTRs and specific RNA-binding proteins, including RNA helicases, in germ granules.

In addition to mRNAs, germ granules contain non-coding RNAs (Table 1). In particular, piRNAs are expected to localize to the granules since these RNAs directly associate with Piwi family proteins found in granules. Also, RNAs from the large and small subunits of mitochondrial ribosomes are germ granule components (Table 1). In the fly, there is evidence that mitochondria-type ribosomes are involved in the 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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES

The detailed pathway of germ granule assembly has not been fully deciphered. Therefore, 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 that determine the fate of germ granules and help to maintain their integrity.

Germ Granule Assembly in the Drosophila Oocytes' Germ Plasm

Drosophila germ granule assembly in the 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). A recent study has identified an osk gene in insect species beyond flies and other dipterans and concluded that the evolutionary origin of osk in insects has been correlated with the appearance of the germ plasm (Lynch et al., 2011).

The correct localization of osk mRNA relies on the organization and reorganization of microtubules in the egg chamber during early oogenesis (Steinhauer and Kalderon, 2006). In stage 2–6 oocytes, microtubules expand their plus-ends into the nurse cells via the ring canals 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 (e.g., grk mRNA) synthesized by nurse cells to the posterior pole of the transcriptionally inactive oocyte (Theurkauf et al., 1992; Saunders and Cohen, 1999; Grieder et al., 2000). After stage 6, these germline microtubules undergo rearrangement triggered by signaling between the oocyte and 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, 2002; Steinhauer and Kalderon, 2006). At stages 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 Kinesin I-dependent transport, dictating the location of germ plasm formation (Ephrussi and Lehmann, 1992; Brendza et al., 2000). 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 de-repression of osk mRNA (Becalska and Gavis, 2010). The resulting Osk protein then initiates germ plasm assembly by recruiting other germ plasm components (e.g., Vas and Tud) to the posterior pole. In particular, the direct interaction between Osk and Vas has been experimentally verified and recognized as an initial step in germ plasm assembly (Breitwieser et al., 1996).

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 (Benton and St Johnston, 2003; Becalska and Gavis, 2010). Therefore, Baz might exert its influence by controlling microtubule polarity to ensure microtubule plus-ends reaching to posterior, thereby restricting Osk-induced germ plasm assembly at the posterior pole (Becalska and Gavis, 2010) (Fig. 1).

image

Figure 1. Illustration of microtubule activity and the localization of osk mRNA and Baz protein during initiation of germ plasm assembly in Drosophila oocyte. During oogenesis stages 2–6, microtubules group the minus-ends in the oocyte and reach the plus-ends into nurse cells. In stages 6–7, microtubules appear from the entire oocyte cortex and are rearranged so that the plus-ends cluster towards the center of the oocyte, along with the disassembly of early stage microtubule organizing center (MTOC). At stages 8–9, 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 translational de-repression is initiated by the accumulation of microtubule plus-ends. Then, the resulting Osk protein starts germ plasm assembly by recruiting other germ plasm components such as Vas and Tud (Ephrussi and Lehmann, 1992; Breitwieser et al., 1996; Steinhauer and Kalderon, 2006; Becalska and Gavis, 2010).

Download figure to PowerPoint

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; Mahowald, 2001; Kloc et al., 2004; Pepling et al., 2007; Strome and Lehmann, 2007; Marlow and Mullins, 2008). Although the assembly machinery of the Balbiani body germ plasm has not been fully elucidated, recent research on the Balbiani body in zebrafish identified the bucky ball (buc) gene as a key germ plasm inducer that governs germ plasm assembly in the Balbiani body (Marlow and Mullins, 2008; Bontems et al., 2009). 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 such as 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 approaches, 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 mRNA of dazl, vas, and nos (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; thus, the hypothesized mechanism is that Buc aggregates germ plasm components already present in the early embryo and prevents their degradation during embryogenesis (Bontems et al., 2009).

image

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).

Download figure to PowerPoint

P granule 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 germ line helicase (GLH) 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 via their 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).

image

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

Download figure to PowerPoint

Worm GLH-1, GLH-2, and GLH-4 proteins contain N-terminal phenylalanine-glycine (FG) repeat domains, which are likely to interact to establish a hydrophobic network of filaments that mimic the role of FG repeats in nuclear pore complex (NPC) proteins (Ribbeck and Gorlich, 2002; Patel et al., 2007; Updike et al., 2011). Work done on GLH proteins revealed 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 (Ribbeck and Gorlich, 2002; Pappu et al., 2008; Hanazawa et al., 2011; Updike et al., 2011). Furthermore, GLH-1 has been suggested to retain the perinuclear localization of PGL proteins and to help lower the saturation point of free PGL proteins in the germline, thereby favoring 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).

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 like liquid droplets, bearing a viscosity roughly 1,000× 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 the anterior and condensation towards the posterior in C. elegans embryos (Brangwynne et al., 2009).

The Role of Tudor and Piwi Proteins in Germ Granule Assembly

The presence of scaffold factors during the assembly of germ granules has been reported in different organisms, for example, Tud domain-containing proteins in the fly, zebrafish, and mouse (Arkov et al., 2006; Vasileva et al., 2009; Huang et al., 2011). As outlined above, Tud domain-containing proteins frequently have multiple Tud domains (Chen et al., 2011). In particular, the fly Tud protein plays an important role in germ granules by binding to sDMAs of the Piwi protein Aubergine (Aub) (Nishida et al., 2009; Kirino et al., 2010). The co-crystallization of heterologously expressed Tud domain 11 of the Tud protein and sDMA-containing Aub peptide revealed the recognition nature of the 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, 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).

image

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).

Download figure to PowerPoint

GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES

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, 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); and 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 found in neoblasts (Reddien et al., 2005; Palakodeti et al., 2008; 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 in neurons. In mice, for example, a particular piRNA and Miwi protein has been shown to colocalize in hippocampal neurons, controlling dendritic spine morphogenesis (Lee et al., 2011). Furthermore, in neurons of Aplysia, specific piRNAs associate with Piwi, and the piRNA–Piwi complex down-regulates 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. Recent work on the cell-free formation of RNP granules provided convincing evidence supporting a prion-like mechanism of 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 that might also be the general principle guiding the assembly of 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 that 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, self-sustainability, and transmissibility, all of which are canonical characteristics of prion-like proteins (Si et al., 2003; Alberti et al., 2009). 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 nervous 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) that 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).

image

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. Conversion to the third state, however, is irreversible and can be pathogenic (Han et al., 2012; Kato et al., 2012).

Download figure to PowerPoint

CONCLUSIONS AND FUTURE OUTLOOK

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES

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, as well as providing insight into 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 (e.g., Vas or Vas homologs), they differ by primary amino acid sequences and are 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 as well as the role of Tud–Piwi–piRNA complexes in the protection of germline DNA from transposons (Table 1). In addition, details of molecular recognition of Piwi proteins' 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 that these assemblies play important roles in stem cell maintenance and memory formation. 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.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES

We thank all members of Arkov laboratory and Chris Trzepacz for critical reading of the manuscript. Work in the A.L.A. laboratory is supported by NSF CAREER award # MCB-1054962 and by NIH grant award R15GM087661 from the National Institute of General Medical Sciences.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. COMPOSITION AND FUNCTION OF GERM GRANULES
  5. ASSEMBLY AND SHAPING OF THE GERM GRANULES
  6. GERM GRANULE-SIMILAR STRUCTURES IN SOMATIC CELLS
  7. CONCLUSIONS AND FUTURE OUTLOOK
  8. ACKNOWLEDGMENTS
  9. REFERENCES