The F-box protein FOG-2 binds the C. elegans multi-ubiquitin chain binding protein-1 (Mcb1) homolog to promote spermatogenesis in the hermaphrodite

Sudhir Nayak1, Edward T Kipreos2, Tim Schedl1
1
Dept of Genetics, Washington University School of Med., St. Louis, MO 63110
2
Dept of Cellular Biology, University of Georgia, Athens, GA 30602

In the C. elegans hermaphrodite, first sperm then oocytes are produced within the same somatic gonad. Based on genetic and molecular analysis, the genes gld-1 and fog-2 are required to promote male sex determination in the hermaphrodite germ line by the down regulation of tra-2, thus allowing for a transient period of spermatogenesis (Schedl and Kimble, 1988; Francis et al., 1995; Jan et al., 1999; Clifford et al., 2000). GLD-1 directly binds the tra-2 3’UTR causing its translational repression. The FOG-2 protein binds GLD-1 and is associated with the tra-2 3’UTR in a ternary complex, but unlike GLD-1, FOG-2 does not make direct contact with the tra-2 mRNA.

The FOG-2 protein contains two domains; an N-terminal F-box motif and a novel C-terminal 200 amino acid putative protein-protein interaction domain. Traditionally, F-box motif containing proteins have been associated with the ubiquitin mediated degradation pathway. Canonical F-box proteins interact with Skp1, a core component of SCF (skp1/cdc53/F-box, E3 ubiquitin ligase) complex, via their N-terminal F-box and recruit specific substrates for ubiquitin modification via their C-terminal protein-protein interaction domains.

The functional definition of an F-box requires that it contain the minimal sequence elements required to interact with the SCF core component Skp1. Therefore, we are conducting a directed screen of all Skp1 homologs in the C. elegans genome using FOG-2 as bait. Interestingly, FOG-2 is able to bind F46A9.5 and F46A9.4, which share the highest overall identity to the defining member yeast Skp1 (44% and 39% respectively). This suggests that the F-box motif in FOG-2 is functional and therefore could play a role in the ubiquitin mediated degradation of specific substrates involving the SCF and 26S proteasome. However, RNAi phenotypes of the aforementioned Skp1 homologs were embryonic lethal so that FOG-2 related functions in sex determination remain unclear.

To define the broad scope of FOG-2 interactions, a C. elegans two-hybrid cDNA library (R. Barstead) was screened. The C. elegans Mcb1 homolog was identified as a FOG-2 interacting protein. RNAi analysis of C. elegans Mcb1 resulted in feminization of the hermaphrodite germ line and partial suppression of the fem-3(q20gf) phenotype, which is analogous to what is observed for gld-1 and fog-2 lf mutations. This provides in vivo support for an interaction between C. elegans Mcb1 and FOG-2. In S. cerevisiae, Mcb1 is a nonessential protein that co-purifies with the 19S regulatory cap of the 26S proteasome, binds multiubiquitin chains in vitro, and plays an ancillary role in substrate-specific protein turnover (van Nocker et al., 1996).

While the yeast Mcb1 protein has been implicated in protein turnover, the C. elegans Mcb1 homolog is unlikely to be involved in GLD-1 degradation as, similar to FOG-2, the Mcb1 homolog appears to promote hermaphrodite spermatogenesis. When RNAi of the Mcb1 homolog is performed in a strain carrying GLD-1::GFP the feminizing phenotype is recapitulated but changes the GLD-1 accumulation or degradation pattern are not observed. Thus, the C. elegans Mcb1 homolog promotes hermaphrodite spermatogenesis and may represent another component of the FOG-2/GLD-1/tra-2 3'UTR translational control complex.

We propose two working models for FOG-2 as a scaffold in recruiting proteins such as Mcb1 into the framework of GLD-1 binding the tra-2 3'UTR and mediating its translational regulation: 1) FOG-2, perhaps in concert with Mcb1, degrades a positive regulator of tra-2 translation. 2) FOG-2 and/or Mcb1 are involved in the ubiquitin modification, but not degradation, of another component(s) of the tra-2 3'UTR ternary complex. A role for F-box proteins in the direct translational regulation of a specific mRNA represents a novel function for this family of proteins.

2001 International Worm Meeting abstract 991

RAS/MAP kinase signaling in meiotic prophase progression

Min-Ho Lee1, Mitsue Ohmachi1, Eric Lambie2, Ross Francis1, Tim Schedl1
1
Dept. of Genetics, Washington University School of Medicine, St. Louis MO
2
Dept. of Biological Sciences, Dartmouth College, Hanover NH

A RAS/MAP kinase signal transduction pathway is essential for meiotic prophase progression in hermaphrodites and males (Church et al., 1994). Strong loss-of-function (lf) mutations in let-60 RAS, lin-45 RAF, mek-2 MAPKK, and mpk-1 MAPK result in germ cells arrested in pachytene. This suggests that the RAS/MAP kinase cascade transduces a signal necessary for progression through pachytene and/or for the transition from pachytene to diplotene/diakinesis. To further understand germline RAS/MAP kinase signaling, we used an antibody that specifically recognizes only the doubly phosphorylated active form of MPK-1 in western analysis as well as in germline staining of wild type, lf and gain-of-function (gf) mutants in this pathway.

MPK-1 has two isoforms, 55 KD and 45 KD; the 55 KD isoform has 68 amino acids more at the N-terminus (Wu and Han, 1994; Lackner and Kim, 1998) and is expressed only in the germline, whereas the 45 KD isoform is expressed in the germline and in the soma. In strong lf mutants of let-60 RAS and mek-2 MAPKK, the doubly phosphorylated active MPK-1 is absent or severely reduced. In wild type hermaphrodite germlines, MPK-1 is doubly phosphorylated and becomes active in two sets of cells: pachytene stage germ cells and in the most proximal diakinesis stage oocytes. Active MPK-1 first appears in the middle of the pachytene region and continues until the end of the pachytene region. To determine whether activation of MPK-1 is sufficient for progression through pachytene or for the transition from pachytene to diplotene/diakinesis, we employed the let-60 (ga89) RAS gf mutantion (Eisenmann and Kim, 1997), which causes early activation of MPK-1 in pachytene stage germ cells. The early activation of MPK-1 in let-60 (ga89) RAS gf mutants is not sufficient to drive germ cells into diplotene earlier than in wild type. This suggests that RAS/MAP kinase signaling may be necessary for progression of germ cells from an early to a late stage of pachytene, while the transition from pachytene to diplotene is controlled by a separate process. Alternatively, the transition from pachytene to diplotene may require not only RAS/MAP kinase signaling but also the execution of additional events such as the completion of homologous recombination. However, the activation of MPK-1 and the relative proportion of pachytene stage germ cells in spo-11 and mrt-2 mutants (Dernburg et al., 1998; Gartner et al., 2000), which never initiate homologous recombination and are defective in monitoring homologous recombination, respectively, are not significantly different from wild type. This suggests that the completion of homologous recombination is not necessary for MPK-1 activation, or for the transition from pachytene to diplotene/diakinesis in C. elegans.

The degree of phosphorylation of MPK-1 decreases rapidly as germ cells make the transition from pachytene to diplotene/diakinesis. This shift in phosphorylation may be important for oocyte growth, because oocyte size is much smaller in let-60 (ga89) RAS gf mutants, which exhibit hyper-activation of MPK-1 in developing oocytes.

The reactivation of MPK-1 in the most proximal oocytes is dependent on an MSP signal from sperm (Miller et al., 2001), suggesting that MAP kinase signaling may function in C. elegans oocyte meiotic maturation (McCarter et al., 1999), as it does in other organisms. Consistent with this possibility, time lapse video analysis of conditional mpk-1 (ga111) mutants shows defects in maturation/ovulation.

2001 International Worm Meeting abstract 256

Identification and characterization of multiple mRNA targets of GLD-1, an RNA binding protein required for germ cell development

Min-Ho Lee1, Rueyling Lin2, Tim Schedl1
1
Dept. of Genetics, Washington University School of Medicine, St. Louis MO
2
Dept. of Mol. Biology and Oncology, U. T. Southwestern Medical Center, Dallas TX

GLD-1, a KH motif containing RNA binding protein in C.elegans, is a germline specific tumor suppressor that is essential for oocyte development. GLD-1 also functions to promote male sex determination in the hermaphrodite germline and has a redundant function to initiate meiotic development. GLD-1 is abundant in the cytoplasm of early meiotic prophase germ cells in the distal region but absent in developing oocytes in the proximal region. Therefore GLD-1 is thought to regulate the translation and/or the stability of a subset of maternal mRNAs that may have functions in oocyte differentiation, maturation/ovulation and early embryogenesis.

We have identified multiple in vivo mRNA targets of GLD-1 by their ability to interact with GLD-1 in cytosol extract. These target mRNAs are preferentially expressed in the germline and as expected, several of them have essential functions in oocyte differentiation, maturation/ovulation and early embryogenesis. Interestingly, subsets of 3 gene families are identified as mRNA targets of GLD-1; chitin binding domain containing, puf-5/-6/-7/-10 and oma-1/-2 TIS11 zinc finger containing. In each subset, single RNAi has no detectable phenotype while double RNAi results in a germline or early embryonic phenotype, suggesting that GLD-1 binds and co-regulates functionally redundant homologs.

Analysis of three mRNA targets (rme-2, oma-1 and oma-2), reveals that GLD-1 acts as a translational repressor. Antibody staining of wild-type hermaphrodite germline shows that the corresponding proteins for three mRNA targets are absent from the distal region where GLD-1 is abundant, while they increase in abundance in growing oocytes in the proximal region where GLD-1 levels fall precipitously. Consistent with GLD-1 functioning as a translational repressor, they prematurely accumulate in the distal region of gld-1 null hermaphrodites. These data imply that GLD-1 is likely acting as a translational repressor for most mRNA targets. However, two mRNA targets, including T23G11.2, are unstable in gld-1 null animals, suggesting that GLD-1 binds and stabilizes them. Upon careful inspection, T23G11.2 mRNA has two small upstream open reading frames in 5’-UTR and therefore is likely a target of nonsense mediated mRNA decay (NMD). In situ staining shows that the level of T23G11.2 mRNA is high in the distal region of wild-type but is very low in developing oocytes in the proximal region. In contrast, T23G11.2 mRNA accumulates to high levels in developing oocytes of smg-2 mutants, which lack NMD. In addition, T23G11.2 mRNA is undetectable in gld-1 null germlines but is present in gld-1 null smg-2 double mutant germlines. GLD-1 thus functions to bind and protect T23G11.2 mRNA from NMD, presumably by repressing translation.

At least four GLD-1 binding sites have been identified so far in three mRNA targets. They are located in 5’-UTR, 5’-end of ORF, and 3-UTR, suggesting that GLD-1 can bind to 5’-end, 3’-end, or both ends depending on the mRNA. For rme-2 mRNA, GLD-1 binds specifically to both the 5’- and the 3’-ends. While a missense mutation (q361) in the invariant GXXG residues inside the KH motif completely abolishes the RNA binding activity of GLD-1, another missense mutation (q126) in the conserved C-terminal region flanking the KH motif only affects binding to the previously identified target mRNA tra-2 (Jan et al., 1999). This suggests that GLD-1 could have more than one binding specificity or binding could be modified by the formation of complexes with other RNA binding proteins, which could allow GLD-1 to control diverse mRNAs.

2001 International Worm Meeting abstract 300

Cloning and characterization of ced-12/elmo, a novel member of the CrkII/Dock180/Rac1 pathway, which is required for phagocytosis and cell migrations

J.M. Kinchen1,2,7, E. Brugnera3, T.L. Gumienny1,2, A. Tosello-Trampont3, L.B. Haney3, K. Nishiwaki4, S. Walk3, R. Francis5,6, T. Schedl6, Y. Qin1, L. Van Aelst1,2, K.S. Ravichandran3, M.O. Hengartner1,2
1
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Habor, NY 11743, USA
2
Department of Molecular Genetics and Microbiology, SUNY-Stony Brook, Stony Brook, NY 11743
3
Beirne B. Carter Center for Immunology Research and Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA
4
PRESTO, Japan Science and Technology Corporation and Fundamental Research Laboratories, NEC Corporation, 34 Miyukigaoka, Tsukuba 305-8501, Japan
5
Exelexis Pharmaceuticals, Inc., P.O. Box 511, South San Francisco, CA 94083, USA
6
Department of Genetics, Washington University School of Medicine, Campus Box 8232, 4566 Scott Avenue, St. Louis, MO 63110, USA.
7
e-mail: kinchen@cshl.org

Programmed cell death (apoptosis) plays a crucial role in animal development as well as in the elimination of damaged cells. The pathway underlying this process is evolutionarily conserved from worms to humans. An important step in apoptosis is the removal of dying cells from the organism via phagocytosis. In C. elegans, removal of dying cells proceeds via two partially redundant pathways. The first pathway is composed of two proteins at the cell membrane, CED-1/SREC and CED-7/ABC1, which may function in the recognition of the dying cell, and CED-6, a putative adapter protein. The second pathway is comprised of CED-2/CrkII, CED-5/Dock180, and CED-10/Rac1, thought to be involved in reorganization of actin filaments, which is required to extend the engulfing cell's membrane around the dying cell.

We genetically and molecularly characterized a novel member of the ced-2/ced-5/ced-10 pathway, ced-12. In C. elegans, CED-12 is required for the engulfment of dying cells and for various cell migrations. Positional cloning of ced-12 revealed that this gene encodes a protein of 731 amino acids, the molecular function of which is unclear from the sequence analysis. However, ced-12 is clearly conserved through evolution, possessing at least one homologue in Drosophila, and two in mice and humans. We have named the mammalian ced-12 orthologues elmo (genes involved in engulfment and motility), and refer to the products of these two ced-12 orthologues as ELMO1 and ELMO2.

In mammalian cells, ELMO-1 functions together with CrkII and Dock180 upstream of Rac1 during phagocytosis. ELMO-1 physically interacts with Dock180 and forms a ternary complex with CrkII, which appears necessary for the functional synergy between the two proteins during phagocytosis. ELMO-1 also regulates Rac-1 dependent cytoskeletal changes and localizes to membrane ruffles. Expression of ELMO-1 or ELMO-2 in CED-12 deficient worms significantly rescues the gonadal migration defect.

These studies show that ced-12/elmo is an evolutionarily conserved upstream regulator of ced-10/Rac1 that affects engulfment and cell migration in both C. elegans and mammalian cells.

2001 International Worm Meeting abstract 64

Multi-pathway Regulation of Meiotic Entry

Dave Hansen1, E. Jane Albert Hubbard2, Pawel Pasierbek3, Josef Loidl3, Tim Schedl1
1
Dept. of Genetics, Washington University School of Med., St. Louis, MO 63110
2
Dept. of Biology, New York University, New York, NY, 10003
3
Dept. of Cytology and Genetics, University of Vienna, 1030 Wien, Austria

At the distal end of the late-larval and adult C. elegans gonad, a population of proliferating germ cells serve as stem cells for potentially hundreds of gametes. As cells move proximally, away from the influence of the Distal Tip Cell (DTC), they leave the mitotic cell cycle and enter meiotic prophase. GLP-1/Notch receptors in distal germ cells are activated by a signal from the DTC, promoting the proliferative state. Gain-of-function (gf) consitutively active alleles of glp-1 cause germ cells to remain proliferative, thereby forming a germline tumor. gld-1 and gld-2 function redundantly to inhibit proliferation and/or promote entry into meiosis and are negatively regulated by the glp-1 pathway (Kadyk and Kimble, 1998; Francis et al. 1995). Animals lacking gld-1 and gld-2 form a germline tumor similar to that seen with a glp-1(gf) allele.

We have employed two genetic screens to identify other genes that are involved in regulating the switch from mitosis to meiosis. One screen identified mutations that enhance a weak glp-1(gf) allele, (oz112 oz120). This screen identified teg-1 (tumorous enhancer of glp-1(gf)), which encodes a 353 amino acid protein with similarity to a human protein that interacts with the T-lymphocyte receptor, CD2. The other screen identified mutations that are synthetic tumorous with gld-2. From this, we obtained multiple alleles of syt-1 (synthetic tumorous), which we cloned and identified as the nos-3 gene (Kraemer et al 1999). This screen also identified another allele of teg-1, demonstrating that teg-1 is synthetic tumorous with gld-2. Neither teg-1 nor nos-3 mutants are tumorous on their own. Thus, teg-1, nos-3 and gld-1 appear to each function redundantly with gld-2 to inhibit proliferation and/or to promote entry into meiosis.

Close examination of the germline tumors in gld-2; teg-1, gld-2; nos-3 and gld-2 gld-1 animals, however, revealed that none are equivalent to the glp-1(oz112gf) tumor, even though the mutations in these four loci appear to be null. Using two markers (anti-REC-8 that stains chromosomes of proliferative nuclei when specific staining conditions are used and anti-HIM-3 (Zetka et al 1999) that stains chromosomes of meiotic nuclei), we have shown that each of these synthetic tumors contain meiotic nuclei while the glp-1(oz112gf) tumors do not. These results suggest that these four genes (gld-1, gld-2, teg-1 and nos-3) could be involved in regulating the progression of meiotic prophase rather than entry into meiotic prophase. Thus the germline tumor would result from germ cells returning to mitosis after failing to progress through meiotic prophase, analogous to the gld-1 single in female germ cells (Francis et al 1995). Conversely, if these genes do act to promote meiotic entry, the presence of meiotic nuclei in the tumors suggests that a loss of gld-2 and gld-1 (or gld-2 and teg-1 or nos-3) is not equivalent to constitutive activation of GLP-1, indicating that there is additional genetic complexity in this process. To distinguish between these two models, we used the weak glp-1 gf allele ar202. At 15C the germ lines of glp-1(ar202) animals are essentially wild type, while at higher temperatures animals display a 'Pro' phenotype (proliferative germ cells in the proximal end of the gonad; see abstract by Pepper, Lo and Hubbard) and a late onset tumorous phenotype (the size of the distal mitotic zone is expanded). We tested gld-1, gld-2 and teg-1 for their ability to enhance the late onset tumorous phenotype of glp-1(ar202) at 15C. We reasoned that if a gene were normally required to promote entry into meiosis, loss of its activity could enhance the late-onset tumorous phenotype of glp-1(ar202). Conversely, if no enhancement of glp-1(ar202) is seen, the gene might not be involved in regulating meiotic entry, but rather meiotic progression.

Mutations in gld-1, gld-2 and teg-1 each enhance the late onset tumorous phenotype of glp-1(ar202) at 15C, supporting the hypothesis that these genes regulate entry into meiosis. Furthermore, there are far more meiotic nuclei in the gld-2 gld-1 and gld-2; teg-1 doubles than in the gld-2 gld-1; teg-1 triple mutant. This suggests that each gene functions to promote entry into meiosis, perhaps through three separate pathways. Since some evidence of meiotic nuclei still exists in the triple mutant, a possible fourth pathway could be involved.

2001 International Worm Meeting abstract 800

A second ksr-like gene in C. elegans: Is it pex-1?

Meera Sundaram1, Christian Rocheleau1, Mitsue Ohmachi2, Tim Schedl2, Diane Church3, Eric Lambie3
1
Dept of Genetics, University of Pennsylvania School of Medicine, Philadelphia PA
2
Dept. of Genetics, Washington University School of Medicine, St. Louis MO
3
Dept. of Biology, Dartmouth College, Hanover NH

KSR (Kinase Suppressor of Ras) is a conserved, Raf-related protein that positively regulates Ras signaling. Drosophila KSR (DKSR) is essential for Ras signaling (Dksr and Ras1 mutants have similar lethal phenotypes; Therrien et al. 1995), whereas C. elegans KSR-1 is a non-essential positive regulator of Ras signaling (ksr-1 mutants have only a few weakly penetrant let-60 Ras-like defects; Kornfeld et al. 1995; Sundaram and Han 1995). Recently, the C. elegans genome sequencing consortium identified a second ksr-like sequence corresponding to the predicted gene F58D5.4, which we refer to as ksr-2. RNA-mediated interference (RNAi) of ksr-2 causes no obvious somatic defects in wild-type, but causes nearly complete rod-like larval lethality in a ksr-1 mutant background. Thus, ksr-1 and ksr-2 are redundant for viability and may together be essential for Ras signaling.

Ras signaling also functions in C. elegans germline development, being necessary for pachytene progression (Church et al. 1995). Mutations in the gene pex-1 display a pachytene arrest phenotype similar to that of let-60 Ras mutants, but do not exhibit either the rod-like larval lethality or vulvaless phenotypes. pex-1 genetically maps in the vicinity of ksr-2 and ksr-2(RNAi) in wild-type causes a sterile phenotype resembling that seen in let-60 Ras and pex-1 mutants. Experiments are underway to determine if ksr-2 and pex-1 are the same gene. If so, then germline ksr activity may be provided exclusively by ksr-2.

2001 International Worm Meeting abstract 757

The C. elegans ptc and ptr genes: A cholesterol connection?

Patricia E. Kuwabara1, Olivier Zugasti1, Min-Ho Lee2, Tim Schedl2, Gregory Jefferis3
1
The Sanger Centre, Wellcome Trust Genome Campus, Hinxton UK CB10 1SA
2
Department of Genetics, Washington University School of Medicine St Louis, MO 63110
3
Stanford University School of Medicine, Stanford, CA 94305-5404

Patched (PTC) is a multipass membrane protein controlling cell fate and proliferation; in humans, PTCH functions as a tumour suppressor. Biochemical analyses have shown that PTC is a receptor for Hedgehog (Hh). It was postulated that PTC and the serpentine membrane protein Smoothened (SMO) form a complex whereby Patched inhibits SMO; Hh relieves this inhibition by binding to PTC. In turn, SMO activates the transcriptional regulator Ci to express TGF-beta and Wnt family members.

In C. elegans, BLAST and Clustal W analyses indicate that there are 3 ptc genes and 26 ptc-related (ptr) genes. Moreover, the hydropathy plots of ptc and ptr genes are very similar; these proteins are predicted to encode 12-pass membrane proteins with topologies similar to those of Patched proteins identified in other organisms. The PTC and PTR proteins also belong to a larger family of proteins containing sterol sensing domains (SSD); members of the SSD family include PTC, Dispatched (CHE-14), HMG CoA reductase, SCAP, SREBP and NPC1.

We have searched the C. elegans genome for other components of the Hh/PTC pathway. There are no obvious Hedgehog or Smoothened homologues encoded by the genome although a large family of Hedgehog-like proteins have been identified (Aspöck et al.1999. Genome Res. 9: 909), some of which may have signalling properties. Moreover, the activity of TRA-1, the single C. elegans homologue of Ci, appears to have been usurped by the sex determination pathway. The apparent absence of many of the components of the Hedgehog/Patched signalling pathway in C. elegans has led us to examine the role of Ce-ptc-1 in the development of the worm. From an evolutionary standpoint, a study of the Ce-ptc genes might shed light on the ancestral roles of Patched proteins or perhaps uncover new functions.

Results of RNAi and mutational deletion studies will be presented indicating that ptc-1 is an essential germline gene (Kuwabara et al. 2000, Genes Dev.14:1933). Animals lacking ptc-1 activity are essentially sterile with multinucleate germ cells arising from a probable defect in germline cytokinesis. The membranes normally separating individual germ cell nuclei are absent in ptc-1 mutants; the loss of these membranes allow multiple nuclei to cycle synchronously through mitosis. We conclude that these membranes maintain autonomous domains within the germline syncytium. It is unclear whether ptc-1 mutants display cytokinesis defects because the cleavage furrow is not formed or because it is not stabilized. Anti-PTC-1 polyclonal antibodies indicate that PTC-1 protein is normally enriched at the apices of the membranes separating individual germ cells. One interpretation of the ptc-1 mutant phenotype is that ptc-1 normally plays a germline-specific role in membrane trafficking. In addition to our studies on the ptc genes, we are undertaking a global analysis of the 26 C. elegans ptr genes. Preliminary results of these studies will be presented.

Taken together, our analyses of the ptc-1 and ptr genes combined with studies of other C. elegans SSD protein encoding genes, such as che-14 (Michaux et al. 2000. Curr. Biol. 10:1098) and npc-1 and npc-2 (Sym et al. 2000. Curr. Biol. 10:527) indicate that the SSD proteins are involved in a diverse range of developmental processes, which may all share a common link to cholesterol.