Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.     

The STAR protein, GLD-1, is a translational regulator of sexual identity in Caenorhabditis elegans.

The Caenorhabditis elegans sex determination gene, tra-2, is translationally regulated by elements in the 3'-untranslated region called TGEs. TGEs govern the translation of mRNAs in both invertebrates and vertebrates, indicating that this is a highly conserved mechanism for controlling gene activity. A factor called DRF, found in worm extracts binds the TGEs and may be a repressor of translation. Using the yeast three-hybrid screen and RNA gel shift analysis, we have found that the protein GLD-1, a germline-specific protein and a member of the STAR family of RNA-binding proteins, specifically binds to the TGEs. GLD-1 is essential for oogenesis, and is also necessary for spermatogenesis and inhibition of germ cell proliferation. Several lines of evidence demonstrate that GLD-1 is a translational repressor acting through the TGEs to repress tra-2 translation. GLD-1 can repress the translation of reporter RNAs via the TGEs both in vitro and in vivo, and is required to maintain low TRA-2A protein levels in the germline. Genetic analysis indicates that GLD-1 acts upstream of the TGE control. Finally, we show that endogenous GLD-1 is a component of DRF. The conservation of the TGE control and the STAR family suggests that at least a subset of STAR proteins may work through the TGEs to control translation.     

Shape-specific nucleotide binding of single-stranded RNA by the GLD-1 STAR domain

Proteins containing the STAR RNA-binding domain fulfill vital roles in RNA biogenesis, yet a detailed understanding of STAR domain RNA binding specificity is lacking. In Caenorhabditis elegans, the STAR protein GLD-1 directly binds the 28 nucleotide recognition element TGE within the 3' untranslated region of tra-2 mRNA. The GLD-1:TGE interaction promotes translational silencing of tra-2 mRNA, marking a pivotal event in the spermatogenesis to oogenesis switch in C.elegans hermaphrodites. By measuring the binding affinities of both GLD-1 and TGE mutants, we have explored the molecular determinants of STAR domain specificity. Site-directed GLD-1 mutants were guided by sequence homology with human splicing factor 1 (SF1), for which an RNA:protein complex structure is available in the work done by Liu et al. The RNA binding affinity of 11 mutant GLD-1 proteins was measured, and their binding specificity was assessed with a series of TGE RNAs containing natural or modified nucleotides. This combinatorial analysis of both RNA and protein mutants revealed a diverse array of specificities of individual nucleotide-binding pockets along the interface. At nucleotide position 18, adenosine appears to be specified by the overall shape of a pocket lined with aliphatic side-chains. At position 19, the high preference for cytidine is dependent on both the length of an amino acid side-chain and the identity of terminal functional groups. The nucleotide 21 binding pocket exhibits low discrimination for cytidine, and accommodates most nucleobases. The highly hydrophobic binding interface and apparent small number of hydrogen bonding read-out interactions at these positions is consistent with our finding that few amino acids seem to function individually in establishing binding specificity. Rather, specificity is conferred by the shape of the nucleotide-binding pocket. Our data provide the first detailed, quantitative analysis of the STAR domain, and highlight features of STAR:RNA recognition that are distinct among single-stranded RNA-binding proteins.     

The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo

During cell-cell fusion, two cells' plasma membranes merge, allowing the cytoplasms to mix and form a syncytium. Little is known about the mechanisms of cell fusion. Here, we asked whether eff-1, shown previously to be essential for fusion in Caenorhabditis elegans, acts directly in the fusion machinery. We show that expression of EFF-1 transmembrane protein drives fusion of heterologous cells into multinucleate syncytia. We obtained evidence that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing. Furthermore, syncytiogenesis requires EFF-1 in both fusing cells. To test whether this mechanism also applies in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion requires EFF-1 in both cells. Thus, although EFF-1-mediated fusion shares characteristics with viral and intracellular fusion, including an apparent hemifusion step, it differs from these reactions in the homotypic organization of the fusion machinery.     

A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific activity of a regulatory hierarchy: At early stages of wild-type development, lin-14 and lin-28 inhibit lin-29 and thus prevent switching. Later, lin-4 inhibits lin-14 and lin-28, allowing activation of lin-29, which in turn triggers the switch in the L4 stage. lin-29 may activate the L/A switch by regulating genes that control cell division, differentiation, and stage-specific gene expression in hypodermal cells.     

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.     

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.     

Conservation of the C.elegans tra-2 3'UTR translational control.

The Caenorhabditis elegans sex-determination gene, tra-2, is translationally regulated by two 28 nt elements (DREs) located in the 3'UTR that bind a factor called DRF. This regulation requires the laf-1 gene activity. We demonstrate that the nematode Caenorhabditis briggsae tra-2 gene and the human oncogene GLI are translationally regulated by elements that are functionally equivalent to DREs. Here, we rename the DREs to TGEs (tra-2 and GLI elements). Similarly to the C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs repress translation of a reporter transgene in a laf-1 dependent manner. Furthermore, they regulate poly(A) tail length and bind DRF. We also find that the C.elegans TGEs control translation and poly(A) tail length in C.briggsae and rodent cells. Moreover, these same organisms contain a factor that specifically associates with the C.elegans TGEs. These findings are consistent with the TGE control being present in C.briggsae and rodent cells. Three lines of evidence indicate that C.briggsae tra-2 and GLI are translationally controlled in vivo by TGEs. First, like C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs control translation and poly(A) tail lengths in C.briggsae and rodent cells, respectively. Second, the same factor in C.briggsae and mammalian cells that binds to the C.elegans tra-2 TGEs binds the C.briggsae tra-2 and GLI TGEs. Third, deletion of the GLI TGE increases GLI's ability to transform cells. These findings suggest that TGE control is conserved and regulates the expression of other mRNAs.     

Lipophilic regulator of a developmental switch in Caenorhabditis elegans

Abstract In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulin-like and transforming growth factor beta (TGFbeta)-like signalling pathways. It has been proposed that lipophilic hormones act downstream of these pathways to regulate dauer formation. One likely target for such a hormone is DAF-12, an orphan nuclear hormone receptor that mediates these developmental decisions and also influences adult lifespan. In order to find lipophilic hormones we have generated lipophilic extracts from mass cultures of C. elegans and shown that they rescue the dauer constitutive phenotype of class 1 daf-2 insulin signalling mutants and the TGFbeta signalling mutant daf-7. These extracts are also able to rescue the lethal dauer phenotype of daf-9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF-12 ligand; extracts, however, have no effect on a DAF-12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF-9 dependent as extracts from a daf-9;daf-12 double mutant do not exhibit this activity. Preliminary fractionation of the lipophilic extracts shows that the activity is hydrophobic with some polar properties, consistent with a small lipophilic hormone. We propose that the dauer rescuing activity is a hormone synthesized by DAF-9 that acts through DAF-12.     

GLD-3 and control of the mitosis/meiosis decision in the germline of Caenorhabditis elegans

Germ cells can divide mitotically to replenish germline tissue or meiotically to produce gametes. In this article, we report that GLD-3, a Caenorhabditis elegans Bicaudal-C homolog, promotes the transition from mitosis to meiosis together with the GLD-2 poly(A) polymerase. GLD-3 binds GLD-2 via a small N-terminal region present in both GLD-3S and GLD-3L isoforms, and GLD-2 and GLD-3 can be co-immunoprecipitated from worm extracts. The FBF repressor binds specifically to elements in the gld-3S 3′-UTR, and FBF regulates gld-3 expression. Furthermore, FBF acts largely upstream of gld-3 in the mitosis/meiosis decision. By contrast, GLD-3 acts upstream of FBF in the sperm/oocyte decision, and GLD-3 protein can antagonize FBF binding to RNA regulatory elements. To address the relative importance of these two regulatory mechanisms in the mitosis/meiosis and sperm/oocyte decisions, we isolated a deletion mutant, gld-3(q741), that removes the FBF-binding site from GLD-3L, but leaves the GLD-2-binding site intact. Animals homozygous for gld-3(q741) enter meiosis, but are feminized. Therefore, GLD-3 promotes meiosis primarily via its interaction with GLD-2, and it promotes spermatogenesis primarily via its interaction with FBF.     

Resonance assignments of the microtubule-binding domain of the C. elegans spindle and kinetochore-associated protein 1

During mitosis, kinetochores coordinate the attachment of centromeric DNA to the dynamic plus ends of microtubules, which is hypothesized to pull sister chromatids toward opposing poles of the mitotic spindle. The outer kinetochore Ndc80 complex acts synergistically with the Ska (spindle and kinetochore-associated) complex to harness the energy of depolymerizing microtubules and power chromosome movement. The Ska complex is a hexamer consisting of two copies of the proteins Ska1, Ska2 and Ska3, respectively. The C-terminal domain of the spindle and kinetochore-associated protein 1 (Ska1) is the microtubule-binding domain of the Ska complex. We solved the solution structure of the C. elegans microtubule-binding domain (MTBD) of the protein Ska1 using NMR spectroscopy. Here, we report the resonance assignments of the MTBD of C. elegans Ska1.     

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.     

The C. elegans SYS-1 protein is a bona fide beta-catenin

C. elegans SYS-1 has key functional characteristics of a canonical beta-catenin, but no significant sequence similarity. Here, we report the SYS-1 crystal structure, both on its own and in a complex with POP-1, the C. elegans TCF homolog. The two structures possess signature features of canonical beta-catenin and the beta-catenin/TCF complex that could not be predicted by sequence. Most importantly, SYS-1 bears 12 armadillo repeats and the SYS-1/POP-1 interface is anchored by a conserved salt-bridge, the "charged button." We also modeled structures for three other C. elegans beta-catenins to predict the molecular basis of their distinct binding properties. Finally, we generated a phylogenetic tree, using the region of highest structural similarity between SYS-1 and beta-catenin, and found that SYS-1 clusters robustly within the beta-catenin clade. We conclude that the SYS-1 protein belongs to the beta-catenin family and suggest that additional divergent beta-catenins await discovery.