C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis

Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.     

The DEAD Box RNA Helicase VBH-1 Is a New Player in the Stress Response in C. elegans

For several years, DEAD box RNA helicase Vasa (DDX4) has been used as a bona fide germline marker in different organisms. C. elegans VBH-1 is a close homolog of the Vasa protein, which plays an important role in gametogenesis, germ cell survival and embryonic development. Here, we show that VBH-1 protects nematodes from heat shock and oxidative stress. Using the germline-defective mutant glp-4(bn2) we found that a potential somatic expression of vbh-1 might be important for stress survival. We also show that the VBH-1 paralog LAF-1 is important for stress survival, although this protein is not redundant with its counterpart. Furthermore, we observed that the mRNAs of the heat shock proteins hsp-1 and sip-1 are downregulated when vbh-1 or laf-1 are silenced. Previously, we reported that in C. elegans, VBH-1 was primarily expressed in P granules of germ cells and in the cytoplasm of all blastomeres. Here we show that during stress, VBH-1 co-localizes with CGH-1 in large aggregates in the gonad core and oocytes; however, VBH-1 aggregates do not overlap with CGH-1 foci in early embryos under the same conditions. These data demonstrate that, in addition to the previously described role for this protein in the germline, VBH-1 plays an important role during the stress response in C. elegans through the potential direct or indirect regulation of stress response mRNAs.     

Genome-wide analysis of germ cell proliferation in C.elegans identifies VRK-1 as a key regulator of CEP-1/p53

Proliferating germ cells in Caenorhabditiselegans provide a useful model system for deciphering fundamental mechanisms underlying the balance between proliferation and differentiation. Using gene expression profiling, we identified approximately 200 genes upregulated in the proliferating germ cells of C. elegans. Functional characterization using RNA-mediated interference demonstrated that over forty of these factors are required for normal germline proliferation and development. Detailed analysis of two of these factors defined an important regulatory relationship controlling germ cell proliferation. We established that the kinase VRK-1 is required for normal germ cell proliferation, and that it acts in part to regulate CEP-1(p53) activity. Loss of cep-1 significantly rescued the proliferation defects of vrk-1 mutants. We suggest that VRK-1 prevents CEP-1 from triggering an inappropriate cell cycle arrest, thereby promoting germ cell proliferation. This finding reveals a previously unsuspected mechanism for negative regulation of p53 activity in germ cells to control proliferation.     

Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.     

Arabidopsis Decapping 5 Is Required for mRNA Decapping,P-Body Formation,and Translational Repression during Postembryonic Development

Eukaryotic processing bodies (P-bodies) are implicated in mRNA storage and mRNA decapping. We previously found that a decapping complex comprising Decapping 1 (DCP1), DCP2, and Varicose in Arabidopsis thaliana is essential for postembryonic development, but the underlying mechanism is poorly understood. Here, we characterized Arabidopsis DCP5, a homolog of human RNA-associated protein 55, as an additional P-body constituent. DCP5 associates with DCP1 and DCP2 and is required for mRNA decapping in vivo. In spite of its association with DCP2, DCP5 has no effect on DCP2 decapping activity in vitro, suggesting that the effect on decapping in vivo is indirect. In knockdown mutant dcp5-1, not only is mRNA decapping compromised, but the size of P-bodies is also significantly decreased. These results indicate that DCP5 is required for P-body formation, which likely facilitates efficient decapping. During wild-type seed germination, mRNAs encoding seed storage proteins (SSPs) are translationally repressed and degraded. By contrast, in dcp5-1, SSP mRNAs are translated, leading to accumulation of their products in germinated seedlings. In vitro experiments using wheat germ extracts confirmed that DCP5 is a translational repressor. Our results showed that DCP5 is required for translational repression and P-body formation and plays an indirect role in mRNA decapping.     

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.     

Highlights from this issue

Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development.     

P-bodies and their functions during mRNA cell cycle: mini-review

P-bodies (processing bodies) are observed in different organisms such as yeast, Caenorhabditis elegans and mammals. A typical eukaryotic cell contains several types of spatially formed granules, such as P-bodies, stress granules and a variety of ribonucleoprotein bodies. These microdomains play important role in mRNA processing, including RNA interference, repression of translation and mRNA decay. The P-bodies components as well as stress granules may play an important role in host defense against viral infection. The complete set of P-bodies protein elements is still poor known. They contain conserved protein core limited to different organisms or to stress status of the cell. P-bodies are related also to some neuronal mRNA granules as well as to maternal RNA granules or male germ cell granules. In this mini-review, we focus on the structure of P-bodies and their function in the mRNA utilization and processing because of the high mRNA's dynamics between different cellular compartments and its key role in modulation of gene expression.     

Automated and customizable quantitative image analysis of whole Caenorhabditis elegans germlines

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: (1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and (2) assessment of these individual nuclei based on their position within the germline. We show the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double-strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields using the C. elegans germline as a model system.     

Novel role of specific Tudor domains in Tudor-Aubergine protein complex assembly and distribution during Drosophila oogenesis

Germ cells give rise to the next generation and contain ribonucleoprotein particles, germ granules. In these granules, Piwi protein Aubergine has been shown to interact with Tudor protein in Drosophila. Tudor protein has 11 Tudor domains and it has been unclear to what extent all these domains are involved in the interaction with Aubergine. Here we present direct biochemical evidence that Tudor-Aubergine interaction surface is composed of different Tudor domains including those that have not been previously implicated in Aubergine recognition. Furthermore, we show that specific single Tudor domains determine localization of Tudor complex to different sites in ovarian germ cells. Our data suggest that multiple Tudor domains of germline proteins from various species are redundantly used for interaction with the same protein partner during germline development.     

Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.     

Cell-nonautonomous inhibition of radiation-induced apoptosis by dynein light chain 1 in Caenorhabditis elegans

The evolutionarily conserved process of programmed cell death, apoptosis, is essential for development of multicellular organisms and is also a protective mechanism against cellular damage. We have identified dynein light chain 1 (DLC-1) as a new regulator of germ cell apoptosis in Caenorhabditis elegans. The DLC-1 protein is highly conserved across species and is a part of the dynein motor complex. There is, however, increasing evidence for dynein-independent functions of DLC-1, and our data describe a novel dynein-independent role. In mammalian cells, DLC-1 is important for cellular transport, cell division and regulation of protein activity, and it has been implicated in cancer. In C. elegans, we find that knockdown of dlc-1 by RNA interference (RNAi) induces excessive apoptosis in the germline but not in somatic cells during development. We show that DLC-1 mediates apoptosis through the genes lin-35, egl-1 and ced-13, which are all involved in the response to ionising radiation (IR)-induced apoptosis. In accordance with this, we show that IR cannot further induce apoptosis in dlc-1(RNAi) animals. Furthermore, we find that DLC-1 is functioning cell nonautonomously through the same pathway as kri-1 in response to IR-induced apoptosis and that DLC-1 regulates the levels of KRI-1. Our results strengthen the notion of a highly dynamic communication between somatic cells and germ cells in regulating the apoptotic process.     

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.