Author Summary Germ cells differ from somatic cells in their unique potential to reproduce a multicellular organism. The immortal germ line links the successive generations in all metazoans, but its development is remarkably diverse. How germline development and survival are regulated in different organisms is far from understood. One funda- mental similarity is the widespread use of post-transcrip- tional mRNA regulation to control the expression of germ cell fate determinants. The development of the C. elegans germ line is a paradigm in the study of translational regulatory networks, composed of conserved RNA-binding or modifying proteins that act as mRNA regulators. Here, we report the discovery of GLS-1, a novel cytoplasmic protein, which we find to form a protein complex with the translational activator GLD-3/Bicaudal-C. This complex promotes and maintains the sperm-to-oocyte switch in hermaphrodites, whereby GLS-1 acts as a molecular mimic of FBF/Pumilio, a translational repressor of sperm promot- ing mRNAs. Furthermore, a GLS-1/GLD-3 complex may also positively regulate mRNAs important for germline survival. Therefore, GLS-1 serves as a new example of how cell fate decisions and tissue development are achieved by modulating the activities of broadly operating translational control networks. lying molecular mechanism is based on multiple interconnected RNA regulators, e.g. Bic-C, PUF, and Nanos proteins, that together comprise a molecular switch to regulate the timely accumulation of first sperm and then oocyte promoting factors. Interestingly, members of these RNA regulatory protein families are broadly conserved and seem to be utilized in other, yet less well understood, cell fate decisions [11]. Two counteracting forces balance the translational output of the key male fate promoting factor, fem-3 [12]; GLD-3L acts as a translational activator whereas two very similar PUF (Pumilio and FBF) proteins, FBF-1 and FBF-2, collectively referred to as FBF, are translational repressors of fem-3 mRNA. FBF-mediated repression of FEM-3 protein synthesis promotes oogenesis indirectly and is aided by a physical interaction with NOS-3, a worm Nanos ortholog [13,14]. Yet to allow sperm production, in males and temporarily in the L4 hermaphrodite larvae, FBF’s oogenesis-promoting activity has to be blocked. This is achieved by zygotic GLD-3L, which reduces FBF’s affinity for its cognate regulatory element in the fem-3 mRNA by binding to FBF’s RNA- binding domain [5]. However, in order to switch to oogenesis, FBF must then be activated by a currently unknown mechanism. These conserved RNA regulators are also involved in the less understood cell fate decision of germ cell survival [11]. In zygotes where GLD-3 is not supplied by the mother, germ cells are correctly specified during embryogenesis but degenerate during postembryonic development. Thus importantly, maternal gld-3 activity is required to prevent germ cell degeneration [5]. Consistent with a role in germline development is that maternal GLD-3 is associated with P granules in the early embryonic P lineage (P1–P4). This also suggests a role for other P granule components in germline maintenance [5]. Additionally, little is known about the roles and interactions of various Nanos and PUF proteins that are required for germline maintenance in C. elegans, Drosophila and mice [13,15–17]. Therefore, we reasoned that further insights into the controls of this cell fate decision might be more easily gained by focusing on the interactions of the single most important player conferring germ cell survival, GLD-3. GLS-1 and Germline Development In this paper, we report the identification and characterization of gls-1 (germline survival defective-1). The gls-1 gene activity is required for multiple aspects of germline development, including the sperm-to-oocyte switch and germline survival. The GLS-1 protein was identified in a yeast two-hybrid screen for GLD-3 interactors, and is a novel P granule component. We demonstrate that during germ cell fate decisions, GLS-1 exerts its roles by modulating GLD-3 activities. We provide evidence that GLS-1 interferes with GLD-3L inhibition of FBF/Pumilio, to assist the translational repression of spermatogenic factors. Furthermore, we find that the maternal GLS-1/GLD-3 complex prevents adult germ cell degeneration. We propose the GLS-1/GLD-3 complex accomplishes this by translationally activating mRNAs encoding survival factors required in the early embryonic germ cell lineage. Results GLS-1 Is a Novel Protein and Expressed in the Germ Line Using the yeast two-hybrid system we aimed to identify new interaction partners of GLD-3. Several partial cDNAs of the gene C36B1.8, which we have named gls-1, were repeatedly found in two independent screens (Text S1). In our analysis of the gls-1 genomic locus we found full-length (fl) gls-1 cDNAs trans-spliced to SL2, suggesting a transcriptional co-regulation with the upstream gene C36B1.7 (Figure 1A,B; Text S1). We termed C36B1.7, dhfr-1, as its predicted ORF encodes a gene similar to dihydrofolate reductase. The gls-1 locus encodes a novel protein with no predictable functional motifs. The amino(N)-terminus of GLS-1 contains two consecutive regions, one rich in serines/threonines/asparagine and one rich in serines/prolines (Figure 1C). The GLD-3 interaction domain is located at the very carboxy(C)-terminus. Although no proteins similar to GLS-1 could be identified in vertebrates we identified the gls-1 locus in C. briggsae and C. remanei where it is also in synteny with the upstream gene dhfr-1 (Text S1). All GLS-1 proteins of the Caenorhabditis clade share approximately 55% amino acid sequence identity when compared to each other. The two shaded regions in the N-terminus in Figure 1C are conserved to almost 80% identity. We conclude that gls-1 is a conserved nematode gene, co-transcribed with dhfr-1. To study the GLS-1 protein, we raised several anti-GLS-1 sera. Two rabbit polyclonal antisera were generated using the central and C-terminal part of GLS-1, respectively (Figure 1C). Both rabbit antisera were affinity purified and recognized a recombi- nant His-epitope tagged GLS-1(FL) of ,150 kDa (Figure 1D). A band of similar size was observed in protein extracts prepared from wild-type L4 animals of either sex but not from gls-1(ef8) deletion mutants, which delete the GLS-1 epitope (Figure 1D). Furthermore, GLS-1 expression is reduced in animals that essentially lack a germ line at 25uC, glp-1(q224ts) and glp-4(bn2ts) (Figure 1D). We conclude that GLS-1 is expressed in the germ line and soma. GLS-1 Is Cytoplasmic and Localizes to P Granules in Germ Cells To address GLS-1 expression in germ cells we performed immunocytochemistry on extruded germ lines. We either used the central rabbit antibody, pre-blocked with gls-1(ef8) protein extract, or a monoclonal anti-GLS-1 antibody, raised against the C- terminus of GLS-1. Germ cells and P granules were visualized with antibodies to the P granule component PGL-1 [18] or GLH-2 [19]. GLS-1 is expressed in all germ cells, with the exception of spermatocytes (not shown). In the adult hermaphrodite, GLS-1 is present in the mitotic region, accumulates during early stages of PLoS Genetics | www.plosgenetics.org 2 May 2009 | Volume 5 | Issue 5 | e1000494