FBF and Its Dual Control of gld-1 Expression in the Caenorhabditis elegans Germline

FBF, a PUF RNA-binding protein, is a key regulator of the mitosis/meiosis decision in the Caenorhabditis elegans germline. Genetically, FBF has a dual role in this decision: it maintains germ cells in mitosis, but it also facilitates entry into meiosis. In this article, we explore the molecular basis of that dual role. Previous work showed that FBF downregulates gld-1 expression to promote mitosis and that the GLD-2 poly(A) polymerase upregulates gld-1 expression to reinforce the decision to enter meiosis. Here we ask whether FBF can act as both a negative regulator and a positive regulator of gld-1 expression and also investigate its molecular mechanisms of control. We first show that FBF co-immunoprecipitates with gld-1 mRNA, a result that complements previous evidence that FBF directly controls gld-1 mRNA. Then we show that FBF represses gld-1 expression, that FBF physically interacts with the CCF-1/Pop2p deadenylase and can stimulate deadenylation in vitro, and that CCF-1 is partially responsible for maintaining low GLD-1 in the mitotic region. Finally, we show that FBF can elevate gld-1 expression, that FBF physically interacts with the GLD-2 poly(A) polymerase, and that FBF can enhance GLD-2 poly(A) polymerase activity in vitro. We propose that FBF can affect polyadenylation either negatively by its CCF-1 interaction or positively by its GLD-2 interaction.     

GLD-3, a bicaudal-C homolog that inhibits FBF to control germline sex determination in C. elegans

The FBF RNA binding proteins control multiple aspects of C. elegans germline development, including sex determination. FBF promotes the oocyte fate at the expense of spermatogenesis by binding a regulatory element in the fem-3 3'UTR and repressing this sex-determining gene. Here we report the discovery of GLD-3, a Bicaudal-C homolog and cytoplasmic protein that physically interacts with FBF. Using RNAi and a gld-3 deletion mutant, we show that GLD-3 promotes the sperm fate, a sex determination effect opposite to that of FBF. By epistasis analysis, GLD-3 acts upstream of FBF, and, in a yeast three-hybrid assay, GLD-3 interferes specifically with FBF binding to the fem-3 3'UTR. We propose that GLD-3 binds FBF and thereby inhibits its repression of target mRNAs.     

The Caenorhabditis elegans homologue of deleted in azoospermia is involved in the sperm/oocyte switch

The Deleted in Azoospermia (DAZ) gene family encodes putative translational activators that are required for meiosis and other aspects of gametogenesis in animals. The single Caenorhabditis elegans homologue of DAZ, daz-1, is an essential factor for female meiosis. Here, we show that daz-1 is important for the switch from spermatogenesis to oogenesis (the sperm/oocyte switch), which is an essential step for the hermaphrodite germline to produce oocytes. RNA interference of the daz-1 orthologue in a related nematode, Caenorhabditis briggsae, resulted in a complete loss of the sperm/oocyte switch. The C. elegans hermaphrodite deficient in daz-1 also revealed a failure in the sperm/oocyte switch if the genetic background was conditional masculinization of germline. DAZ-1 could bind specifically to mRNAs encoding the FBF proteins, which are translational regulators for the sperm/oocyte switch and germ stem cell proliferation. Expression of the FBF proteins seemed to be lowered in the daz-1 mutant at the stage for the sperm/oocyte switch. Conversely, a mutation in gld-3, a gene that functionally counteracts FBF, could partially restore oogenesis in the daz-1 mutant. Together, we propose that daz-1 plays a role upstream of the pathway for germ cell sex determination.     

The C. elegans sex determination protein MOG-3 functions in meiosis and binds to the CSL co-repressor CIR-1

In the germ line of the Caenorhabditis elegans hermaphrodite, nuclei either proliferate through mitosis or initiate meiosis, finally differentiating as spermatids or oocytes. The production of oocytes requires repression of the fem-3 mRNA by cytoplasmic FBF and nuclear MOG proteins. Here we report the identification of the sex determining gene mog-3 and show that in addition to its role in gamete sex determination, it is necessary for meiosis by acting downstream of GLP-1/Notch. Furthermore, we found that MOG-3 binds both to the nuclear proteins MEP-1 and CIR-1. MEP-1 is necessary for oocyte production and somatic differentiation, while the mammalian CIR-1 homolog counters Notch signaling. We propose that MOG-3, MEP-1 and CIR-1 associate in a nuclear complex which regulates different aspects of germ cell development. While FBF triggers the sperm/oocyte switch by directly repressing the fem-3 mRNA in the cytoplasm, the MOG proteins play a more indirect role in the nucleus, perhaps by acting as epigenetic regulators or by controlling precise splicing events.     

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

Analysis of the Multiple Roles of Gld-1 in Germline Development: Interactions with the Sex Determination Cascade and the Glp-1 Signaling Pathway

The Caenorhabditis elegans gene gld-1 is essential for oocyte development; in gld-1 (null) hermaphrodites, a tumor forms where oogenesis would normally occur. We use genetic epistasis analysis to demonstrate that tumor formation is dependent on the sexual fate of the germline. When the germline sex determination pathway is set in the female mode (terminal fem/fog genes inactive), gld-1 (null) germ cells exit meiotic prophase and proliferate to form a tumor, but when the pathway is set in the male mode, they develop into sperm. We conclude that the gld-1 (null) phenotype is cell-type specific and that gld-1 (+) acts at the end of the cascade to direct oogenesis. We also use cell ablation and epistasis analysis to examine the dependence of tumor formation on the glp-1 signaling pathway. Although glp-1 activity promotes tumor growth, it is not essential for tumor formation by gld-1 (null) germ cells. These data also reveal that gld-1 (+) plays a nonessential (and sex nonspecific) role in regulating germ cell proliferation before their entry into meiosis. Thus gld-1 (+) may negatively regulate proliferation at two distinct points in germ cell development: before entry into meiotic prophase in both sexes (nonessential premeiotic gld-1 function) and during meiotic prophase when the sex determination pathway is set in the female mode (essential meiotic gld-1 function).     

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.     

FOG-2, a novel F-box containing protein, associates with the GLD-1 RNA binding protein and directs male sex determination in the C. elegans hermaphrodite germline

Male sex determination in the Caenorhabditis elegans hermaphrodite germline requires translational repression of tra-2 mRNA by the GLD-1 RNA binding protein. We cloned fog-2 by finding that its gene product physically interacts with GLD-1, forming a FOG-2/GLD-1/tra-2 3'untranslated region ternary complex. FOG-2 has an N-terminal F-box and a novel C-terminal domain called FTH. Canonical F-box proteins act as bridging components of the SCF ubiquitin ligase complex; the N-terminal F-box binds a Skp1 homolog, recruiting ubiquination machinery, while a C-terminal protein-protein interaction domain binds a specific substrate for degradation. However, since both fog-2 and gld-1 are necessary for spermatogenesis, FOG-2 cannot target GLD-1 for ubiquitin-mediated degradation. We propose that FOG-2 also acts as a bridge, bringing GLD-1 bound to tra-2 mRNA into a multiprotein translational repression complex, thus representing a novel function for an F-box protein. fog-2 is a member of a large, apparently rapidly evolving, C. elegans gene family that has expanded, in part, by local duplications; fog-2 related genes have not been found outside nematodes. fog-2 may have arisen during evolution of self-fertile hermaphroditism from an ancestral female/male species.     

Cyclin E and Cdk2 control GLD-1, the mitosis/meiosis decision, and germline stem cells in Caenorhabditis elegans

Coordination of the cell cycle with developmental events is crucial for generation of tissues during development and their maintenance in adults. Defects in that coordination can shift the balance of cell fates with devastating clinical effects. Yet our understanding of the molecular mechanisms integrating core cell cycle regulators with developmental regulators remains in its infancy. This work focuses on the interplay between cell cycle and developmental regulators in the Caenorhabditis elegans germline. Key developmental regulators control germline stem cells (GSCs) to self-renew or begin differentiation: FBF RNA-binding proteins promote self-renewal, while GLD RNA regulatory proteins promote meiotic entry. We first discovered that many but not all germ cells switch from the mitotic into the meiotic cell cycle after RNAi depletion of CYE-1 (C. elegans cyclin E) or CDK-2 (C. elegans Cdk2) in wild-type adults. Therefore, CYE-1/CDK-2 influences the mitosis/meiosis balance. We next found that GLD-1 is expressed ectopically in GSCs after CYE-1 or CDK-2 depletion and that GLD-1 removal can rescue cye-1/cdk-2 defects. Therefore, GLD-1 is crucial for the CYE-1/CDK-2 mitosis/meiosis control. Indeed, GLD-1 appears to be a direct substrate of CYE-1/CDK-2: GLD-1 is a phosphoprotein; CYE-1/CDK-2 regulates its phosphorylation in vivo; and human cyclin E/Cdk2 phosphorylates GLD-1 in vitro. Transgenic GLD-1(AAA) harbors alanine substitutions at three consensus CDK phosphorylation sites. GLD-1(AAA) is expressed ectopically in GSCs, and GLD-1(AAA) transgenic germlines have a smaller than normal mitotic zone. Together these findings forge a regulatory link between CYE-1/CDK-2 and GLD-1. Finally, we find that CYE-1/CDK-2 works with FBF-1 to maintain GSCs and prevent their meiotic entry, at least in part, by lowering GLD-1 abundance. Therefore, CYE-1/CDK-2 emerges as a critical regulator of stem cell maintenance. We suggest that cyclin E and Cdk-2 may be used broadly to control developmental regulators.     

Control of the proliferation versus meiotic development decision in the C. elegans germline through regulation of GLD-1 protein accumulation

Maintenance of the stem cell population in the C. elegans germline requires GLP-1/Notch signaling. We show that this signaling inhibits the accumulation of the RNA binding protein GLD-1. In a genetic screen to identify other genes involved in regulating GLD-1 activity, we identified mutations in the nos-3 gene, the protein product of which is similar to the Drosophila translational regulator Nanos. Our data demonstrate that nos-3 promotes GLD-1 accumulation redundantly with gld-2, and that nos-3 functions genetically downstream or parallel to fbf, an inhibitor of GLD-1 translation. We show that the GLD-1 accumulation pattern is important in controlling the proliferation versus meiotic development decision, with low GLD-1 levels allowing proliferation and increased levels promoting meiotic entry.     

C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are well-conserved RNA-binding proteins, which regulate mRNA translation mainly through control of poly(A) elongation. Here, we show that CPB-3, one of the four CPEB homologs in C. elegans, positively regulates multiple aspects of oocyte production. CPB-3 protein was highly expressed in early meiotic regions of the hermaphrodite gonad. Worms deficient in cpb-3 were apparently impaired in germ cell proliferation and differentiation including sperm/oocyte switching and progression of female meiosis. We also show that cpb-3 is likely to promote the meiotic entry in parallel with gld-3, a component of one of the redundant but essential genetic pathways for the entry to and progression through meiosis. Taken together, CPEB appears to have a conserved role in the early phase of meiosis and in the sperm/oocyte specification, in addition to its reported function during meiotic progression.     

Structural basis for the antagonistic roles of RNP-8 and GLD-3 in GLD-2 poly(A)-polymerase activity

Cytoplasmic polyadenylation drives the translational activation of specific mRNAs in early metazoan development and is performed by distinct complexes that share the same catalytic poly(A)-polymerase subunit, GLD-2. The activity and specificity of GLD-2 depend on its binding partners. In Caenorhabditis elegans, GLD-2 promotes spermatogenesis when bound to GLD-3 and oogenesis when bound to RNP-8. GLD-3 and RNP-8 antagonize each other and compete for GLD-2 binding. Following up on our previous mechanistic studies of GLD-2–GLD-3, we report here the 2.5 Å resolution structure and biochemical characterization of a GLD-2–RNP-8 core complex. In the structure, RNP-8 embraces the poly(A)-polymerase, docking onto several conserved hydrophobic hotspots present on the GLD-2 surface. RNP-8 stabilizes GLD-2 and indirectly stimulates polyadenylation. RNP-8 has a different amino-acid sequence and structure as compared to GLD-3. Yet, it binds the same surfaces of GLD-2 by forming alternative interactions, rationalizing the remarkable versatility of GLD-2 complexes.     

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.