Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins

The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germ-cell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes' function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.     

GLH-1, the C. elegans P granule protein, is controlled by the JNK KGB-1 and by the COP9 subunit CSN-5

The GLHs (germline RNA helicases) are constitutive components of the germline-specific P granules in the nematode Caenorhabditis elegans and are essential for fertility, yet how GLH proteins are regulated remains unknown. KGB-1 and CSN-5 are both GLH binding partners, previously identified by two-hybrid interactions. KGB-1 is a MAP kinase in the Jun N-terminal kinase (JNK) subfamily, whereas CSN-5 is a subunit of the COP9 signalosome. Intriguingly, although loss of either KGB-1 or CSN-5 results in sterility, their phenotypes are strikingly different. Whereas csn-5 RNA interference (RNAi) results in under-proliferated germlines, similar to glh-1/glh-4(RNAi), the kgb-1(um3) loss-of-function mutant exhibits germline over-proliferation. When kgb-1(um3) mutants are compared with wild-type C. elegans, GLH-1 protein levels are as much as 6-fold elevated and the organization of GLH-1 in P granules is grossly disrupted. A series of additional in vivo and in vitro tests indicates that KGB-1 and CSN-5 regulate GLH-1 levels, with GLH-1 targeted for proteosomal degradation by KGB-1 and stabilized by CSN-5. We propose the ;good cop: bad cop' team of CSN-5 and KGB-1 imposes a balance on GLH-1 levels, resulting in germline homeostasis. In addition, both KGB-1 and CSN-5 bind Vasa, a Drosophila germ granule component; therefore, similar regulatory mechanisms might be conserved from worms to flies.     

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

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

The GLH proteins,Caenorhabditis elegans P granule components,associate with CSN-5 and KGB-1, proteins necessary for fertility,and with ZYX-1, a predicted cytoskeletal protein

The GLH proteins belong to a family of four germline RNA helicases in Caenorhabditis elegans. These putative ATP-dependent enzymes localize to the P granules, which are nonmembranous complexes of protein and RNA exclusively found in the cytoplasm of all C. elegans germ cells and germ cell precursors. To determine what proteins the GLHs bind, C. elegans cDNA libraries were screened by the yeast two-hybrid method, using GLHs as bait. Three interacting proteins, CSN-5, KGB-1, and ZYX-1, were identified and further characterized. GST pull-down assays independently established that these proteins bind GLHs. CSN-5 is closely related to the subunit 5 protein of COP9 signalosomes, conserved multiprotein complexes of plants and animals. RNA interference (RNAi) with csn-5 results in sterile worms with small gonads and no oocytes, a defect essentially identical to that produced by RNAi with a combination of glh-1 and glh-4. KGB-1 is a putative JNK MAP kinase that GLHs bind. A kgb-1 deletion strain has a temperature-sensitive, sterile phenotype characterized by the absence of mature oocytes and the presence of trapped, immature oocytes that have undergone endoreplication. ZYX-1 is a LIM domain protein most like vertebrate Zyxin, a cytoskeletal adaptor protein. In C. elegans, while zyx-1 appears to be a single copy gene, neither RNAi depletion nor a zyx-1 deletion strain results in an obvious phenotype. These three conserved proteins are the first members in each of their families reported to associate with germline helicases. Similar to the loss of GLH-1 and GLH-4, loss of either CSN-5 or KGB-1 causes oogenesis to cease, but does not affect the initial assembly of P granules.     

PAN-1, a P-granule component important for C. elegans fertility, has dual roles in the germline and soma

In Caenorhabditis elegans, P granules are germline-specific, RNA-containing granules that segregate into the germline precursor cell during early embryogenesis. In this short report, PAN-1, which previously has been found by others in screens for genes causing larval molting defects, is identified here as a novel P-granule component and a binding partner of GLH-1 (Germline RNA Helicase-1), a constitutive, germline-specific, P-granule protein. The PAN-1 predicted protein contains multiple leucine-rich repeats (LRRs) and regions with similarities to F-box proteins. Antibodies raised against PAN-1 reveal it is present both in the soma and the germline. In the germline, PAN-1 uniquely localizes to P granules from the first larval stage onward and is unusual for a P-granule component in lacking recognizable RNA binding motifs. Homozygous pan-1(gk142) deletion worms arrest as larvae that are unable to molt and this phenotype is also seen with pan-1(RNAi) into wild type worms. pan-1(RNAi) into the somatic RNAi-defective strain rrf-1(pk1417) bypasses the larval arrest and allows an assessment of PAN-1 function in the germline. We find pan-1(RNAi) is variably effective in knocking down PAN-1 protein and results in adult progeny that display multiple germline defects. These phenocopies range from under-proliferation of the germline, as also seen with loss of GLH-1, to the induction of endomitotic replication in oocytes, both defects that result in sterility, to fertile animals with significantly reduced progeny numbers. Thus, while loss of PAN-1 in the soma inhibits molting, this report demonstrates that PAN-1 is also a P-granule component that is essential for fertility.     

MEG-1 and MEG-2 are embryo-specific P-granule components required for germline development in Caenorhabditis elegans

In Caenorhabditis elegans, germ granules called P granules are directly inherited from mother to daughter and segregate with the germ lineage as it separates from the soma during initial embryonic cell divisions. Here we define meg-1 and meg-2 (maternal-effect germ-cell defective), which are expressed in the maternal germline and encode proteins that localize exclusively to P granules during embryonic germline segregation. Localization of MEG-1 to P granules depends upon the membrane-bound protein MES-1. meg-1 mutants exhibit multiple germline defects: P-granule mis-segregation in embryos, underproliferation and aberrant P-granule morphology in larval germ cells, and ultimately, sterility as adults. The penetrance of meg-1 phenotypes increases when meg-2 is also absent. Loss of the P-granule component pgl-1 in meg-1 mutants increases germ-cell proliferation, while loss of glh-1 decreases proliferation. Because meg-1 is provided maternally but its action is required in the embryonic germ lineage during segregation from somatic lineages, it provides a critical link for ensuring the continuity of germline development from one generation to the next.     

Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans

We report that four putative germline RNA helicases, GLHs, are components of the germline-specific P granules in Caenorhabditis elegans. GLH-3 and GLH-4, newly discovered, belong to a multi-gene glh family. Although GLHs are homologous to Drosophila VASA, a polar granule component necessary for oogenesis and embryonic pattern formation, the GLHs are distinguished by containing multiple CCHC zinc fingers. RNA-mediated interference (RNAi) reveals the GLHs are critical for oogenesis. By RNAi at 20 degrees C, when either loss of GLH-1 or GLH-4 alone has no effect, loss of both GLH-1 and GLH-4 results in 97% sterility in the glh-1/4(RNAi) offspring of injected hermaphrodites. glh-1/4(RNAi) germlines are under-proliferated and are without oocytes. glh-1/4(RNAi) animals produce sperm; however, spermatogenesis is delayed and the sperm are defective. P granules are still present in glh-1/4(RNAi) sterile worms as revealed with antibodies against the remaining GLH-2 and GLH-3 proteins, indicating the GLHs function independently in P granule assembly. These studies reveal that C.elegans can use GLH-1 or GLH-4 to promote germline development.     

Bioinformatic analysis of P granule-related proteins: insights into germ granule evolution in nematodes

Germ cells in many animals possess a specialized cytoplasm in the form of granules that contain RNA and protein complexes essential for the function and preservation of the germline. The mechanism for the formation of these granules is still poorly understood; however, the lack of conservation in their components across different species suggests evolutionary convergence in the assembly process. Germ granules are assumed to be present in all nematodes with a preformed germline. However, few studies have clearly identified these structures in species other than Caenorhabditis elegans and even less have carried functional analysis to provide a broader panorama of the granules composition in the phylum. We adopted a bioinformatics approach to investigate the extension of conservation in nematodes of some known C. elegans germ granule components, as a proxy to understand germ granules evolution in this phylum. Unexpectedly, we found that, in nematodes, the DEAD box RNA helicase Vasa, a conserved protein among different phyla, shows a complex history of clade-specific duplications and sequence divergence. Our analyses suggest that, in nematodes, Vasa’s function might be shared among proteins like LAF-1, VBH-1, and GLH-1/-2/-3 and GLH-4. Key components of P granules assembly in C. elegans, like the PGL protein family, are only preserved in Caenorhabditis species. Our analysis suggests that germ granules assembly may not be conserved in nematodes. Studies on these species could bring insight into the basic components required for this pathway.     

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly.     

P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA

The germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA.     

The Caenorhabditis elegans eukaryotic initiation factor 5A homologue, IFF-1, is required for germ cell proliferation, gametogenesis and localization of the P-granule component PGL-1

Eukaryotic initiation factor 5A (eIF-5A) was originally isolated as a translation initiation factor. However, this function has since been reconsidered, with recent studies pointing to roles for eIF-5A in mRNA metabolism and trafficking [Microbiol. Mol. Biol. Rev. 66 (2002) 460; Eur. Mol. Biol. Org. J. 17 (1998) 2914]. The Caenorhabditis elegans genome contains two eIF-5A homologues, iff-1 and iff-2, whose functions in vivo were examined in this study. The iff-2 mutation causes somatic defects that include slow larval growth and disorganized somatic gonadal structures in hermaphrodites. iff-2 males show disorganized tail sensory rays and spicules. On the other hand, iff-1 mRNA is expressed in the gonad, and the lack of iff-1 activity causes sterility with an underproliferated germline resulting from impaired mitotic proliferation in both hermaphrodites and males. In spite of underproliferation, meiotic nuclei are observed, as revealed by presence of immunoreactivity to the anti-HIM-3 antibody; however, no gametogenesis occurs in the iff-1 gonads. These phenotypes are in part similar to the mutants affected in the components of P granules, which are the C. elegans counterparts of germ granules [Curr. Top Dev. Biol. 50 (2000) 155]. We found that localization of the P-granule component PGL-1 to P granules is disrupted in the iff-1 mutant. In summary, the two C. elegans homologues of eIF-5A act in different tissues: IFF-2 is required in the soma, and IFF-1 is required in the germline for germ cell proliferation, for gametogenesis after entry into meiosis, and for proper PGL-1 localization on P granules.     

The DEAD box RNA helicase VBH-1 is required for germ cell function in C. elegans

Vasa and Belle are conserved DEAD box RNA helicases required for germ cell function. Homologs of this group of proteins in several species, including mammals, are able to complement a mutation in yeast (DED1) suggesting that their function is highly conserved. It has been proposed that these proteins are required for mRNA translation regulation, but their specific mechanism of action is still unknown. Here we describe functions of VBH-1, a C. elegans protein closely related to Belle and Vasa. VBH-1 is expressed specifically in the C. elegans germline, where it is associated with P granules, the C. elegans germ plasm counterpart. vbh-1(RNAi) animals produce fewer offspring than wild type because of defects in oocyte and sperm production, and embryonic lethality. We also find that VBH-1 participates in the sperm/oocyte switch in the hermaphrodite gonad. We conclude that VBH-1 and its orthologs may perform conserved roles in fertility and development.     

The PGL family proteins associate with germ granules and function redundantly in Caenorhabditis elegans germline development

PGL-1 is a constitutive protein component of C. elegans germ granules, also known as P granules. Maternally supplied PGL-1 is essential for germline development but only at elevated temperature, raising the possibility that redundant factors provide sufficient function at lower temperatures. We have identified two PGL-1-related proteins, PGL-2 and PGL-3, by sequence analysis of the C. elegans genome and by a yeast two-hybrid screen for proteins that interact with PGL-1. PGL-3 is associated with P granules at all stages of development, while PGL-2 is associated with P granules only during postembryonic development. All three PGL proteins interact with each other in vitro. Furthermore, PGL-1 and PGL-3 are co-immunoprecipitated from embryo extracts, indicating that they are indeed in the same protein complex in vivo. Nevertheless, each PGL protein localizes to P granules independently of the other two. pgl-2 or pgl-3 single-mutant worms do not show obvious defects in germline development. However, pgl-1; pgl-3 (but not pgl-2; pgl-1) double-mutant hermaphrodites and males show significantly enhanced sterility at all temperatures, compared to pgl-1 alone. Mutant hermaphrodites show defects in germline proliferation and in production of healthy gametes and viable embryos. Our findings demonstrate that both PGL-2 and PGL-3 are components of P granules, both interact with PGL-1, and at least PGL-3 functions redundantly with PGL-1 to ensure fertility in both sexes of C. elegans.     

Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex

Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.     

Germ plasm in Caenorhabditis elegans, Drosophila and Xenopus

Special cytoplasm, called germ plasm, that is essential for the differentiation of germ cells is localized in a particular region of Caenorhabditis elegans, Drosophila and Xenopus eggs. The mode of founder cell formation of germline, the origin and behavior of the germline granules, and the molecules localized in germline cells are compared in these organisms. The common characteristics of the organisms are mainly as follows. First, the founder cells of germline are established before the intiation of gastrulation. Second, the germline granules or their derivatives are always present in germline cells or germ cells throughout the life cycle in embryos, larvae, and adults. Lastly, among the proteins localized in the germ plasm, only Vasa protein or its homolog is detected in the germline cells or germ cells throughout the life cycle. As the protein of vasa homolog has been reported to be also localized in the germline-specific structure or nuage in some of the organisms without the germ plasm, the possibility that the mechanism for differentiation of primordial germ cells is basically common in all organisms with or without the germ plasm is discussed.     

An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans

Control of gene expression at the translational level is crucial for many developmental processes. The mRNA cap-binding protein, eIF4E, is a key player in regulation of translation initiation; appropriate levels of eIF4E are essential for normal cell-cycle regulation and tissue differentiation. The observation that eIF4E levels are elevated during gametogenesis in several organisms suggests that eIF4E might have a specific role in gamete formation as well. We show that one of the five isoforms of C. elegans eIF4E, IFE-1, is enriched in the germline and is a component of germ granules (P granules). The association of IFE-1 with P granules requires the P-granule protein PGL-1. In vitro PGL-1 interacts directly with IFE-1, but not with the other four isoforms of eIF4E. Analysis of animals depleted of IFE-1 by RNAi shows that IFE-1 is required for spermatogenesis, specifically for efficient progression through the meiotic divisions and for the production of functional sperm, in both hermaphrodites and males. The requirement for IFE-1 is highly sensitive to temperature. IFE-1 is not required for oogenesis, as ife-1(RNAi) hermaphrodites produce viable progeny when normal sperm are supplied. Consistent with a primary role in spermatogenesis, ife-1 mRNA levels are highest in regions of the gonad undergoing spermatogenesis. Our results suggest that C. elegans spermatogenesis requires either this specific isoform of eIF4E or an elevated level of eIF4E.     

Analysis of RNA associated with P granules in germ cells of C. elegans adults

P granules are cytoplasmic structures of unknown function that are associated with germ nuclei in the C. elegans gonad, and are localized exclusively to germ cells, or germ cell precursors, throughout the life cycle. All the known protein components of P granules contain putative RNA-binding motifs, suggesting that RNA is involved in either the structure or function of the granules. However, no specific mRNAs have been identified within P granules in the gonad. We show here that P granules normally contain a low level of RNA, and describe conditions that increase this level. We present evidence that several, diverse mRNAs, including pos-1, mex-1, par-3, skn-1, nos-2 and gld-1 mRNA, are present at least transiently within P granules. In contrast, actin and tubulin mRNA and rRNA are either not present in P granules, or are present at relatively low levels. We show that pgl-1 and the glh (Vasa-related) gene family, which encode protein components of P granules, do not appear essential for RNA to concentrate in P granules; these proteins may instead function in events that are a prerequisite for RNAs to be transported efficiently from the nuclear surface.     

A novel function for the Sm proteins in germ granule localization during C. elegans embryogenesis

General mRNA processing factors are traditionally thought to function only in the control of global gene expression. Here we show that the Sm proteins, core components of the splicesome, also regulate germ granules during early C. elegans development. Germ granules are large cytoplasmic particles that localize to germ cells and their precursors during embryogenesis of diverse organisms. In C. elegans, germ granules, called P granules, are segregated to the germline precursor cells during embryogenesis by asymmetric cell division, and they remain in germ cells at all stages of development. We found that at least some Sm proteins are components of P granules. Moreover, disruption of Sm activity caused defects in P granule localization to the germ cell precursors during early embryogenesis. In contrast, loss of other splicing factor activities had no effect on germ granule control in the embryo. These observations suggest that the Sm proteins control germ granule integrity and localization in the early C. elegans embryo and that this role is independent of pre-mRNA splicing. Thus, a highly conserved splicing factor may have been adapted to control both snRNP biogenesis and the localization of components important for germ cell function.