Title: The STAR protein, GLD-1, is a translational regulator of sexual identity in Caenorhabditis elegans
Abstract: Article4 January 1999free access The STAR protein, GLD-1, is a translational regulator of sexual identity in Caenorhabditis elegans Eric Jan Eric Jan Department of Cell and Molecular Biology and Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, 60611 USA Search for more papers by this author Cynthia K. Motzny Cynthia K. Motzny Search for more papers by this author Laura E. Graves Laura E. Graves Department of Cell and Molecular Biology and Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, 60611 USA Search for more papers by this author Elizabeth B. Goodwin Corresponding Author Elizabeth B. Goodwin Department of Cell and Molecular Biology and Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, 60611 USA Search for more papers by this author Eric Jan Eric Jan Department of Cell and Molecular Biology and Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, 60611 USA Search for more papers by this author Cynthia K. Motzny Cynthia K. Motzny Search for more papers by this author Laura E. Graves Laura E. Graves Department of Cell and Molecular Biology and Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, 60611 USA Search for more papers by this author Elizabeth B. Goodwin Corresponding Author Elizabeth B. Goodwin Department of Cell and Molecular Biology and Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, 60611 USA Search for more papers by this author Author Information Eric Jan1,‡, Cynthia K. Motzny2,‡, Laura E. Graves1 and Elizabeth B. Goodwin 1 1Department of Cell and Molecular Biology and Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, 60611 USA 2Roosevelt University, School of Science and Mathematics, Albert A.Robin Campus, 1651 McConnor Parkway, Schaumburg, IL, 60173-4348 USA ‡E.Jan and C.K.Motzny contributed equally to this work The EMBO Journal (1999)18:258-269https://doi.org/10.1093/emboj/18.1.258 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The precise temporal and spatial expression of key regulatory genes is crucial for normal development. It is now apparent that translational control by elements in the 3′-untranslated region (3′-UTR) play major roles in regulating developmentally important genes (Wickens et al., 1996). For example, elements in the 3′-UTR of the Drosophila hunchback and oskar mRNAs are necessary for repressing translation and hence controlling anterior–posterior axis formation (Wharton and Struhl, 1991; Kim-Ha et al., 1995; Rongo et al., 1995). While many 3′-UTR cis-acting elements are known, only a few trans-acting factors have been identified. As a result, the mechanisms underlying 3′-UTR translational controls are poorly understood. To comprehend these mechanisms better, we sought trans-acting factors that interact with the translational regulatory elements in the 3′-UTR of the Caenorhabditis elegans sex-determining gene, tra-2. Caenorhabditis elegans has two sexes: hermaphrodite and male. Hermaphrodites are essentially female in the soma but make both sperm and oocytes in the germline. The primary signal for sex determination is the ratio of the number of X chromosomes to sets of autosomes, such that animals with two X chromosomes (XX) develop as hermaphrodites while animals with a single X chromosome (XO) develop as males (for a review see Meyer, 1997). The X to autosomal ratio controls the activity of a number of genes that act in a cascade to regulate sexual cell identity (Figure 1; see Meyer, 1997). Figure 1.Genetic control of sex determination in C.elegans. For simplicity, genes that act early to control both sex determination and dosage compensation are omitted (for a review see Meyer, 1997). (A) Sex determination in somatic tissues. Eight genes are critical determinants of somatic sexual fates: her-1, three tra genes, three fem genes and laf-1. In XO animals, her-1 and laf-1 inhibit tra-2; consequently, the fem genes inhibit tra-1 and male development ensues. In XX animals, her-1 is not active and tra-3 represses laf-1 activity; therefore, tra-2 inhibits the fem genes allowing tra-1 to promote female development. In addition, tra-1 may feed back positively on to tra-2 to amplify commitment to female development (Okkema and Kimble, 1991). (B) Sex determination in the germline. Seven of the genes that regulate somatic sexual fate also play a major role in regulation of germline sexual identity: her-1, laf-1, tra-2, tra-3 and the fem genes. In addition, three fog genes (Schedl and Kimble, 1988; Barton and Kimble, 1990; Ellis and Kimble, 1995) and six mog genes (Graham and Kimble, 1993; Graham et al., 1993) affect germline but not somatic sexual fates. In XO animals, her-1 and laf-1 inhibit tra-2, permitting fog-1, fog-3 and the fem genes to direct spermatogenesis. The XX germline is more complex because first sperm and then oocytes are made. The her-1, fog-2 and laf-1 genes are thought to repress tra-2 to promote spermatogenesis; then after a brief period of spermatogenesis, the mog genes repress male-determining genes so that oogenesis can proceed. In contrast to the soma, tra-1 is not the terminal regulator in germline sex determination. Download figure Download PowerPoint The sex-determining gene, tra-2, is required for female cell fates (Hodgkin and Brenner, 1977). tra-2 is predicted to encode a large transmembrane protein, called TRA-2A, that is necessary to inhibit downstream male determinants (Kuwabara et al., 1992). TRA-2A is thought to be part of a signal transduction pathway that is important in ensuring that all the cells in an animal adopt the same sexual fate (Kuwabara et al., 1992). In males, TRA-2A activity is low and male development ensues (Hodgkin, 1980). Proper male development requires that tra-2 activity is repressed. Dominant gain-of-function (gf) mutations have been identified that result in excessive tra-2 activity causing inappropriate female development in both XX and XO animals. XX animals develop as females (they make no sperm) and XO animals produce oocytes in the germline and yolk in the intestine (Doniach, 1986). The gf mutations all map to a direct repeat located in the tra-2 3′-UTR (Goodwin et al., 1993). This direct repeat consists of two regulatory elements, called TGEs (for tra-2 and GLI elements), which control tra-2 activity by repressing the translation of the tra-2 mRNA (Goodwin et al., 1993; Jan et al., 1997). In addition, the TGEs bind a factor, called DRF (direct repeat factor), that is present in crude worm extracts. Previous analyses suggest that DRF is a repressor of translation (Goodwin et al., 1993; Jan et al., 1997). Our working model is that the binding of DRF to the TGEs results in translational repression of tra-2. TGEs control translation not only in C.elegans, but also in the nematode Caenorhabditis briggsae and mammalian cells. Furthermore, the translation of at least three genes [C.briggsae tra-2, human GLI (Jan et al., 1997) and C.elegans tra-1 (E.Jan, Y.Yoo and E.B.Goodwin, unpublished results)] is controlled by TGEs. These results indicate the TGE control is a conserved mechanism that may regulate the translation of a number of mRNAs. To explore further the mechanism of how TGEs control sexual development by regulating the translation of tra-2, we screened for TGE-binding factors using the yeast three-hybrid system. We found that the protein GLD-1 (defective in germline development) specifically binds to the TGEs. GLD-1 is a member of the STAR (signal transduction and activation of RNA) family of RNA-binding proteins which are present in both invertebrates and vertebrates and are essential for many developmental decisions (Vernet and Artzt, 1997). The RNA targets of STAR proteins and how STAR proteins regulate RNA activity are poorly understood. Here, we show that GLD-1 is a translational repressor that acts through the TGEs to inhibit tra-2 translation. The finding that the TGE control is a conserved mechanism raises the possibility that other STAR family members may act via TGEs to regulate translation. Results Identification of GLD-1 as a TGE-binding factor To understand better the mechanism of the TGE control, we sought factors that bind to the C.elegans tra-2 TGEs. Recently, a yeast three-hybrid screen was developed to identify RNA-binding proteins (SenGupta et al., 1996). The three-hybrid system selects for proteins that bind to specific RNA sequences. A diagram of the yeast three-hybrid screen is shown in Figure 2A. Briefly, a hybrid RNA is expressed that contains the MS2 coat protein-binding site, fused to an RNA ‘bait’, in our case the tra-2 TGEs. For the ‘bait’, both tra-2 TGEs arranged in tandem were used, which is a total of 60 nucleotides (see Materials and methods for sequences). This arrangement is precisely how the TGEs are found in the tra-2(+) 3′-UTR (Goodwin et al., 1993). A fusion protein consisting of the MS2 coat protein and the LexA DNA-binding domain anchors the hybrid RNA to the promoter of either the lacZ or HIS3 reporter gene. The binding of a protein expressed from the cDNA library to the TGEs results in the formation of a tripartite complex that activates the transcription of the reporter genes. Figure 2.Identification of GLD-1 as a TGE-binding factor using the yeast three-hybrid screen. (A) Model of the yeast three hybrid. Four constructs were used (SenGupta et al., 1996). The first construct, which is stably integrated into the yeast genome, consists of the LexA-binding site upstream of the reporter genes, lacZ and HIS3. The second construct expresses a fusion protein of the LexA DNA-binding domain and an MS2 viral coat protein. The MS2 coat protein binds specifically to a 21 nucleotide RNA stem–loop. The third construct expresses an RNA hybrid consisting of two 21 nucleotide stem–loops and the RNA ‘bait’. In this study, the RNA ‘bait’ is the C.elegans tra-2 TGEs (see Materials and methods for sequences). The fourth construct consists of the C.elegans cDNA library fused to the GAL4 DNA activation domain. When a protein (Protein X) expressed by the cDNA library binds the bait RNA, lacZ and HIS3 reporter genes are transcriptionally activated. Colonies are tested for β-gal activity by a color assay and for the ability to grow on plates lacking histidine. (B) Identification of a clone that specifically requires TGEs to activate reporter transgene. Clones that required the RNA hybrid for activation of reporter transgene transcription were tested for RNA-binding specificity with several different RNAs. As shown, the test RNA hybrids carried either the two MS2 target 21 nucleotide stem–loops alone (MS2) or the MS2 stem–loops fused to the TGEs (MS2/tra-2TGE), to a poly(A)30 (MS2/A30) or to an IRE (MS2/IRE). The IRE is found in untranslated regions of mRNAs encoding proteins involved in iron metabolism (for a review, see Rouault and Klausner, 1996). The IRE acts to control RNA translation and stability and is known to bind specifically to the IRE-binding protein. These test hybrids were transformed into yeast that contained the cDNA clones and tested for lacZ and HIS3 expression in the presence of 5 mM aminotriazole. Shown is the only positive clone that activated transcription of lacZ when the RNA hybrid contained the TGEs but failed to activate transcription when the other RNA hybrids were used. The one positive clone was sequenced and was found to code for GLD-1. Download figure Download PowerPoint We used a C.elegans cDNA library to screen for proteins that interact with the tra-2 TGEs. The library was transfected into yeast, and colonies that expressed β-gal and that grew on plates lacking histidine were selected. From a screen of 6×105 transformants, 87 positive colonies were isolated. Of these 87 positives, 20 were dependent on the presence of the hybrid MS2–tra-2 TGE RNA. From these 20, we screened for clones that specifically required the TGEs to activate lacZ and HIS3 reporter genes. Toward this end, we tested the ability of the 20 clones to activate transcription of lacZ and HIS3 when the hybrid RNA contains the TGEs but not when it contains other 3′-UTR elements. Four target hybrid RNAs were used: the MS2-binding site alone or the MS2-binding site fused to the TGEs, to a poly(A)30 or to an iron response element (IRE) (Figure 2B). Of the 20 positives, only one activated transcription when the hybrid bait contained the TGEs but failed to activate transcription when other RNA baits were used (Figure 2B). Sequence analysis of the single positive clone revealed that it coded for the protein GLD-1. GLD-1 is a germline-specific cytoplasmic protein (Jones and Schedl, 1995), and is part of a family of RNA-binding proteins called the STAR family (for review see Vernet and Artzt, 1997). The STAR proteins are thought to link signal transduction pathways and RNA metabolism. The hallmarks of the STAR family are a single KH RNA-binding domain and conserved QUA1 and QUA2 domains. The STAR family includes the murine and human SAM68 (Darnell et al., 1994; Fumagalli et al., 1994; Taylor and Shalloway, 1994) and SF1 (Kramer, 1992; Toda et al., 1994; Agger and Freimuth, 1995; Arning et al., 1996), the murine, Xenopus, Zebrafish and human QUAKINGs (Ebersole et al., 1996; Vernet and Artzt, 1997; Zorn et al., 1997) and the Drosophila HOW/WHO proteins (Baehrecke, 1997; Zaffran et al., 1997). STAR proteins play important roles in a number of developmental events. They are necessary for embryogenesis and myelination in mice, as well as notochord differentiation in Xenopus embryos and muscle development in Drosophila (Ebersole et al., 1996; Zaffran et al., 1997; Zorn and Krieg, 1997). How STAR proteins perform these roles is still poorly understood. GLD-1 has multiple roles in germline development (Francis et al., 1995a). GLD-1 is essential for oogenesis. In gld-1. lf) animals, the oocyte germline fails to progress through meiosis and re-enters mitosis, resulting in overproliferation of germline cells and consequently a tumorous germline (Francis et al., 1995a). GLD-1 has non-essential roles in germline proliferation and sex determination (Francis et al., 1995a,b). With regard to sex determination, GLD-1 is necessary for hermaphrodite spermatogenesis. gld-1. lf) XX animals make few or no sperm. We hypothesize that spermatogenesis results from the repression of tra-2 translation by GLD-1. GLD-1 interacts specifically with TGE To test whether GLD-1 directly interacts with the C.elegans tra-2 TGEs, we used RNA gel mobility assays and asked whether purified bacterially expressed GST–GLD-1 fusion protein bound to the tra-2 TGEs. Incubation of GST–GLD-1 with RNA containing the wild-type tra-2(+) 3′-UTR resulted in a slower migrating band, indicating complex formation (Figure 3A, compare lane 1 with 2–5; Figure 3C). GST–GLD-1 and the C.elegans tra-2 3′-UTR RNA had a binding constant of ∼500 nM. Complex binding was not due to GST since GST alone did not bind RNA (data not shown). The binding of the tra-2 3′-UTR to GLD-1 was dependent upon the TGEs since GST–GLD-1 bound only weakly to mutant tra-2 3′-UTRs in which the TGEs were deleted (Figure 3A, compare lane 6 with 7–9; Figure 3C). We were unable to saturate binding to the mutant tra-2 3′-UTR RNAs, indicating that the binding constant is much greater than 500 nM and is probably due to non-specific binding. We also performed competition experiments and found that the tra-2(+) 3′-UTR but not mutant 3′-UTRs competed for GLD-1 binding to the tra-2(+) 3′-UTR (data not shown). The broadness of the GLD-1 shift with the wild-type tra-2(+) 3′-UTR may be due to oligomerization of the GLD-1 as previous studies have shown that GLD-1 can self-associate (Chen et al., 1997). Figure 3.GLD-1 binds specifically to the TGEs. Binding of GLD-1 to the TGEs was tested by RNA gel mobility shift analysis (Goodwin et al., 1997). (A) A 15 fmol aliquot of 32P-labeled C.elegans tra-2 3′-UTR (lane 1) or mutant (lane 6) 3′-UTR in which the TGEs have been deleted was incubated alone or with increasing amounts of purified bacterially expressed GST–GLD-1 protein (lanes 2–5 and 7–9). The amounts of GST–GLD-1 protein added to the reactions are as follow: lanes 2–5: 0.1, 0.15, 0.2 and 0.25 μg; lanes 7–9: 0.15, 0.2 and 0.25 μg. Reactions were loaded and electrophoresed on a 4% non-denaturing polyacrylamide gel. The gel was dried and autoradiographed. Slower migrating bands represent complex formation (brackets); the faster migrating bands indicate free probe (arrows). (B) A 1 fmol aliquot of 32P-labeled C.briggsae tra-2 TGE (EJ-19) or mutant C.briggsae tra-2 TGE (EJ-32 and EJ-35, see Materials and methods for sequences) was incubated alone (lanes 1, 3 and 5) or with 0.65 μg of GST–GLD-1 (lanes 2, 4 and 6). Slower migrating bands are due to complex formation (arrow); faster migrating bands are indicative of free probe. (C) Summary of GLD-1 binding. Binding to GST–GLD-1 was determined by RNA gel shifts using radiolabeled RNA and by competitive RNA gel shifts. Specific binding was scored positive if labeled RNA bound GST–GLD-1. Competitive RNA gel shifts were performed on full-length wild-type and mutant C.elegans tra-2 3′-UTRs and small RNAs which either contained or did not contain the C.elegans tra-2 TGEs (data not shown). Left: names of RNAs (for sequences, see Materials and methods). Middle: diagrams of RNAs. Black arrows represent Ce-tra-2 TGEs, stippled arrows represent the Cb-tra-2 TGE, and open arrows represent the GLI TGE. The sizes of the deletions are indicated in parentheses. Right: the different RNAs were scored for the ability (+) or inability (−) to bind GLD-1. Asterisks beside arrows indicate mutant sequences. EJ-32 and EJ-35 carry small deletions or point mutations in the TGE that disrupt DRF binding to the C.briggsae tra-2 TGE (unpublished results; for sequences see Materials and methods). (D) The KH domain of GLD-1 is required to bind to the TGEs. A 1 fmol aliquot of 32P-labeled tra-2 3′-UTR was added alone (lane 1) or with 0.25 μg of GST–GLD-1 (lane 2) or 0.25 μg of GST–GLD-1(q361) (lane 3) in which the KH domain contains an amino acid substitution (Gly227→Asp; Jones and Schedl, 1995). Slower migrating bands are due to complex formation (arrow); the faster migrating bands indicate free probe (arrowhead). Non-specific binding is shown by the arrowhead with the asterisk. Download figure Download PowerPoint To explore further the binding specificity of GLD-1, we examined the ability of GLD-1 to bind small RNAs containing just the TGEs (Figure 3B and summarized in C). Similarly to the full-length tra-2 3′-UTRs, radiolabeled RNAs containing just the TGEs (EBG-9) bound GST–GLD-1 but RNAs in which the TGEs had been deleted (EBG-11) did not (Figure 3C). Previously, we identified functional TGEs in the 3′-UTR of the C.briggsae tra-2 and human GLl mRNAs (Jan et al., 1997). One would predict that GLD-1 should also bind these elements. Indeed, we found that GST–GLD-1 specifically associated with small RNAs containing the C.briggsae tra-2 (EJ-19) and GLI (EJ-38) TGEs (Figure 3B, lanes 1 and 2, and data not shown; Figure 3C), but did not form a complex with RNAs that contained a mutant C.briggsae tra-2 TGE (EJ-32 and EJ-35, Figure 3B, lanes 2–6). EJ-35 contains a six nucleotide deletion within the 31 nucleotide C.briggsae tra-2 TGE, and EJ-32 carries the same six nucleotide deletion as well as three base substitutions (see Materials and methods for sequences). In conclusion, GLD-1 binds specifically to TGEs. If GLD-1 regulates tra-2 activity by the TGEs, then previously identified mutations in GLD-1, which dramatically reduce GLD-1 activity, may disrupt the ability of GLD-1 to bind TGEs. Previously, Schedl and colleagues identified a point mutation, called GLD-1(q361) (Gly227 to Asp), in the KH domain that results in a strong loss-of-function phenotype (Jones and Schedl, 1995). gld-1. q361) homozygous animals do not produce sperm, and gld-1. q361)/+ heterozygous animals have a semi-dominant germline phenotype where some of the animals make only oocytes (Francis et al., 1995a). To test whether this point mutation altered the ability of GLD-1 to bind the TGEs, we asked whether GST–GLD-1(q361) mutant protein bound TGEs in a gel mobility shift assay. GST–GLD-1(q361) was not able to form a complex with the wild-type tra-2 3′-UTR (Figure 3D, compare lanes 2 and 3). These results show that the loss-of-function phenotype of GLD-1 correlates with loss of GLD-1 binding to the TGEs, supporting the idea that TGE binding is required for GLD-1 function. GLD-1 is a component of DRF Previous analyses suggest that DRF is a repressor of tra-2 translation (Goodwin et al., 1993). Hence, one would predict that GLD-1 should be a component of DRF. To address this, we compared the migration of DRF in an RNA gel mobility assay in the presence and absence of GLD-1 antibody. Often DRF is a doublet, possibly indicating that it consists of multiple factors. Addition of GLD-1 antibody resulted in a reduction of DRF binding (Figure 4A, compare lanes 2 with 3). GLD-1 is probably a component of both complexes, since addition of antibody reduces both. Pre-absorbed GLD-1 antibody and an antibody to GST do not significantly affect the mobility of the DRF–tra-2 3′-UTR complex, indicating that the inhibition by GLD-1 antibody is specific (Figure 4A, lanes 4 and 5). The antibody results support the idea that GLD-1 is a component of DRF. Figure 4.GLD-1 is a component of DRF. (A) GLD-1 antibodies inhibit DRF activity. To test whether GLD-1 is a component of DRF, GLD-1 antibody was added to C.elegans extract in an RNA gel mobility assay. Radiolabeled RNAs containing the C.elegans tra-2 3′-UTR (lane 1) were incubated with either C.elegans adult extract (lane 2), adult extract and 0.4 μg of GLD-1 antibody (lane 3), adult extract and GLD-1 antibody pre-absorbed to GLD-1 (lane 4) or adult extract and 1 μg of GST antibody (lane 5). Reactions were loaded on a 6% polyacrylamide gel and autoradiographed. Slower migrating bands are DRF complexes (arrows); faster migrating band indicates free probe (arrowhead). (B) GLD-1 polyclonal antibodies are specific to GLD-1. Purified GST–GLD-1 (lane 1), wild-type C.elegans adult extract (lane 2) and mutant C.elegans adult extract from gld-1. q485lf) animals were loaded on an 8% SDS–polyacrylamide gel. Shown is a Western blot using GLD-1 antibody. A single band is detected for GST–GLD-1 (66 kDa) and in C.elegans extract (58 kDa). However, no band is detected in mutant gld-1. q485null) extracts: gld-1. q485lf) animals do not produce GLD-1 protein (lane 3; Jones et al., 1996). Download figure Download PowerPoint GLD-1 represses tra-2 activity via the TGEs in vivo If GLD-1 is important in regulating translation, then it should control the activity of mRNAs that contain TGEs in vivo. To address this, we asked whether the expression of GLD-1 could inhibit the activity of reporter transgenes that carried TGEs. Presently, it is not possible to assay transgenes in the germline of C.elegans. Consequently, we performed this analysis by ectopically expressing GLD-1 in the soma. To express GLD-1 in the soma, a construct containing the heat shock promotor (hsp16-41) fused to the entire GLD-1-coding region (hsp::GLD-1) was made. Four reporter transgenes were used: all coded for the lacZ gene and contained either the wild-type tra-2 3′-UTR [lacZ::tra-2(+)3′UTR], a mutant tra-2 3′-UTR in which one [lacZ::tra-2(−32)3′UTR] or both TGEs [lacZ::tra-2(−60)3′UTR] were removed, or a 108 nucleotide deletion [lacZ::tra-2(−108)3′UTR] that removes the TGEs plus flanking sequences. The use of the lacZ::tra-2(−32)3′UTR transgene is a particularly sensitive assay for regulation, since a single TGE is able to partially repress translation (Goodwin et al., 1997). The transgenes were controlled by the inducible heat shock promoter (hsp16-41). Transgenic animals carrying hsp::GLD-1 and either lacZ::tra-2(+)3′UTR?, lacZ::tra-2(−32)3′UTR, lacZ::tra-2(−60)3′UTR or lacZ::tra-2(−108)3′UTR transgenes were heat shocked and the percentage of transgenic animals with intestinal β-gal staining were scored. We found that ectopic expression of GLD-1 in animals carrying TGEs resulted in a dramatic decrease in intestinal β-gal staining. In the absence of GLD-1, 7 and 59% of transgenic animals carrying lacZ::tra-2(+)3′UTR and lacZ::tra-2(−32)3′UTR, respectively, had β-gal staining in intestinal cells (Figure 5A, Table I). In contrast, when GLD-1 was expressed in the soma, 0% of lacZ::tra-2(+)3′UTR and only 18% of lacZ::tra-2(−32)3′UTR transgenic animals had intestinal β-gal staining (Figure 5B, Table I). Ectopic expression of GLD-1 had little or no effect on the β-gal expression of lacZ::tra-2(−60)3′UTR and lacZ::tra-2(−108)3′UTR (Figure 5C and D, Table I). Figure 5.GLD-1 represses tra-2 activity via the TGEs in vivo. Lateral views of C.elegans adult animals with anterior to the left. The reporter lacZ gene is driven by the C.elegans heat shock promotor (hsp16-41; Stringham et al., 1992) and is fused to the nuclear localization signal, such that β-gal staining is primarily nuclear. Left: different tra-2 3′-UTRs inserted downstream of the lacZ reporter gene. (A and C) Transgenic C.elegans animals carrying the different 3′-UTR reporter transgenes in the absence of GLD-1. (B and D) Transgenic C.elegans animals containing the different reporter transgenes and the hsp::GLD-1 transgene that expresses ectopic GLD-1 in the soma. The hsp::GLD-1 contains the heat shock promotor which drives the expression of GLD-1 from the gld-1 cDNA. (A) Animals carrying lacZ::tra-2(−32)3′UTR in which one TGE is deleted. β-Gal activity is detected in four intestinal cells (arrow). (C) Animals carrying lacZ::tra-2(−60)3′UTR in which both TGEs were deleted. β-Gal activity is detected in 15 intestinal cells (arrow)). When GLD-1 is expressed, there is a decrease in β-gal intestinal staining in C.elegans animals carrying the lacZ::tra-2(−32)3′UTR (B), but not in animals carrying lacZ::tra-2(−60)3′UTR (D). β-Gal activity is detected in 18 intestinal cells (arrow). Download figure Download PowerPoint Table 1. GLD-1 represses the TGE control in vivo Reporter transgenea GLD-1 transgeneb % animals with intestinal β-gal stainingc lacZ::tra-2(+)3′UTR none 7% (n = 59) hsp::GLD-1 0% (n = 26) hsp::GLD-1(q361) 5% (n = 22) lacZ::tra-2(−32)3′UTR none 59% (n = 80) hsp::GLD-1 18% (n = 56) hsp::GLD-1(q361) 60% (n = 52) lacZ::tra-2(-60)3′UTR none 68% (n = 105) hsp::GLD-1 53% (n = 58) hsp::GLD-1(q361) 74% (n = 46) lacZ::tra-2(-108)3′UTR none 52% (n = 82) hsp::GLD-1 48% (n = 33) hsp::GLD-1(q361) n.d. a Reporter transgenes containing the C.elegans heat shock promotor (hsp16-41) upstream of the reporter lacZ gene. The reporter transgenes contain an NLS. Wild-type tra-2 or mutant tra-2 3′-UTRs were inserted downstream of the lacZ gene. In all experiments, adult transgenic worms were heat shocked for 2 h at 33°C and allowed to recover for an additional 2 h at 20°C before being fixed and stained for β-gal activity. b Transgenic C.elegans animals containing different reporter transgenes as shown on the left were crossed into transgenic C.elegans animals containing hsp::GLD-1 or hsp::GLD-1(q361). Both hsp::GLD-1 and hsp::GLD-1(q361) are controlled by the heat shock promotor (hsp16-41) and carry the unc-54 3′-UTR. hsp::GLD-1 contains the coding region for wild-type GLD-1 and hsp::GLD-1(q361) carries the coding region for a mutant GLD-1 in which there is an amino acid substitution from Gly227→Asp. c Transgenic animals were scored as positive if blue precipitate was detectable in intestinal cells at 630× magnification. Intestinal cells were scored since genetic evidence indicates that TGE regulation is present in these cells (Doniach, 1986). Percentiles represent the values of one typical transgenic line. Other lines gave similar results. n = total number of animals scored from at least four different experiments. n.d., not determined. If GLD-1 represses tra-2 translation, then expressio