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Translational control of development inC. elegans
seminars in C E L L & D E V E L OP M E N T A L B I OL OG Y , Vol 8, 1997: pp 551]559
Translational control of development in C. elegans Elizabeth B. GoodwinU and Thomas C. Evans†
Translational control by the 39untranslated regions (39UTRs) of mRNAs contributes to important events throughout the development of C. elegans. In oocytes and early embryos, maternal mRNAs are controlled by 39UTR elements to restrict translation of their protein products to specific blastomeres. Localized translation is probably critical for specifying blastomere identity. In both germline and somatic cells, mRNAs from sex determining genes are translationally repressed by 39UTR controls. These controls balance the activities that specify male and female cell fates. During larval development, the temporal sequence of cell lineages requires 39UTR-mediated regulation of heterochronic genes by a small non-protein coding RNA. We review what is known about these translational control mechanisms in C. elegans. This overview illustrates that translational control by 39UTR elements is a powerful mechanism for regulating the expression of multiple gene products in diverse cell types during development of a multi-cellular animal.
over, it is largely unknown how these mechanisms are connected to other pathways Že.g. signal transduction and cell cycle controls. to regulate developmental decisions. In this review, we discuss the role of translational control in the development of the nematode C. elegans. Work on C. elegans has revealed that 39UTRmediated regulation governs a wide range of developmental events throughout the animal’s life. So far, translational regulation has been implicated in the control of early embryogenesis, sex determination of both germ line and somatic cells and the temporal execution of cell lineages during larval development. Using the genetic, cell biological and molecular approaches available in the worm, cis-acting elements and trans-acting factors that control mRNA translation are being identified and pathways that integrate these factors with development are being discovered. For this discussion, we define translational control somewhat broadly; regulation of translation may occur by a variety of different mechamisms. In most examples of translationally controlled mRNAs in C. elegans, it is not known if 39UTRs control ribosome initiation or elongation. 39UTR elements and transacting factors could directly interact with the translational apparatus, or they could sequester mRNAs away from translation components. Furthermore, in some cases regulation of mRNA transport from the nucleus has not been ruled. Future analysis of the cellular and molecular details will certainly be of interest in resolving these issues.
Key words: translational control r 39UTR r C. elegans r sex determination r heterochronic genes Q1997 Academic Press Ltd
DURING ANIMAL DEVELOPMENT, proteins that govern cell fate are expressed at defined levels, at exact times and in specific cells. This precise control of gene expression is essential to specify when cells divide, where they go and what they become. In both vertebrates and invertebrates, recent work indicates that the control of translation by elements in the 39untranslated region Ž39UTR. of mRNAs is critical to regulating a number of developmental processes Žsee refs. 1 and 2 for reviews.. Yet, the mechanisms of 39UTR-mediated regulation remain obscure. More-
Maternal mRNA regulation and development of the early embryo In C. elegans, as in most animal species, the early development of embryos depends on mRNAs and proteins provided by oocytes. These maternal gene products are transcribed in germ cells and thus must be controlled post-transcriptionally in embryos. Recent work demonstrates that precise regulation of maternal factors is critical to creating distinct identi-
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From the Department of Cell and Molecular Biology and the Lurie Cancer Center, Northwestern University Medical School, 303 East Chicago Ave., Chicago IL 60611 USA and †Department of Cell and Structural Biology, University of Colorado Health Science Center, Denver, CO 80262 USA Q1997 Academic Press Ltd 1084-9521r 97r 060551q 09 $25.00r 0r sr970180
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ceiving and transducing signals from posterior cells.4 One putative GLP-1 ligand, the APX-1 protein, is expressed exclusively in the posterior-most cell of the four-cell embryo, called P2.9 The P2 localization of the APX-1 ligand and the anterior localization of the GLP-1 receptor is probably needed to spatially constrain cell signaling so that the two anterior blastomeres can adopt different cell fates.4 In addition to the localization of these factors, the production of all of these proteins are temporally regulated; proteins are generally not present in oocytes, they begin to appear between the one- and four-cell stages and most factors disappear by the eight- to 24-cell stages. Therefore early C. elegans embryogenesis relies on elaborate temporal and spatial control systems that restrict key factors to specific cells in the embryo. What are the mechanisms that mediate this remarkable regulation? The glp-1, skn-1, apx-1 and pal-1 mRNAs are transcribed in germ cells and are, at least initially, evenly distributed in the cytoplasms of oocytes and all cells of the early embryo.6,7,9,10 Therefore the unique locations of these proteins appear to result from distinct patterns of localized mRNA translation andror protein stability. Reporter RNA experiments showed that the glp-1 39UTR was sufficient to restrict the translation of a heterologous RNA to anterior cells of early embryos, without affecting lo-
ties for the early blastomeres of the embryo. Several observations suggest that the control of maternal mRNA translation plays a prominent role in this process. The first cell division in the embryo is asymmetric, generating an anterior blastomere and a posterior blastomere which differ in composition and developmental potential Žreviewed in refs. 3 and 4..3,4 With each subsequent cell division, each daughter blastomere aquires a unique identity either by inheriting a particular set of factors following asymmetric cell division, or through specific interactions with neighboring cells. Mutant screens have identified several genes whose maternally contributed gene products are critical to the specification of early cell identities. The precise pattern of early embryogensis requires the localization of the protein products of some of these genes to specific blastomeres ŽFigure 1.. By the four-cell stage, the two putative transcription factors SKN-1 and PAL-1 are only present in the posterior cells.5,6 These two proteins appear to function as determinants of posterior cell types;5,6,8 their localization to posterior cells may be critical to restricting posterior fates to these cells.5,6 In contrast, the membrane-bound receptor GLP-1 is expressed only in the two anterior blastomeres at the four-cell stage.7 GLP-1 regulates the determination of anterior cells by re-
Figure 1. A pathway for the localization of maternal factors during early C. elegans embryogenesis. Polarity is created in the 1 cell zygote leading to the posterior localization of P-granules Žsmall dots. and other asymmetries. Anterior Žshaded. and posterior Žunshaded. cells arise following cell division. The membrane proteins GLP-1 Žblack. and APX-1 Žstippled., and the nuclear proteins SKN-1 Žgray. and PAL-1 Žblack. are localized by the four cell stages. The embryo at the far right represents the different identieis for all cells arising from the localization of maternal factors and a cell interaction Žarrow.. Known factors Že.g. PAR proteins. and predicted unknown factors Ž? . that control different aspects of polarity and maternal mRNA regulation are indicated. Some factors are required to promote protein expression Že.g. PIE-1 . while others repress expression in non-expressing cells Že.g. MEX-1 and MEX-3 ..
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calization of the RNA.7 A similar experiment showed that the pal-1 39UTR largely restricts reporter protein expression to posterior cells.6 These results suggest that translational controls via the 39UTRs of glp-1 and pal-1 at least in part create the asymmetric distribution of their protein products. Analyses of how the translation of maternal mRNAs is controlled have only just begun. As described above, the glp-1 39UTR confers regulation on a reporter RNA; translation is repressed in oocytes, is activated in early embryos and is localized to anterior cells.7 A U-rich region at the 39 end is required to repress translation in ooctyes. In contrast, a distinct region is required for repressing translation in posterior cells, but is not required for oocyte repression. Therefore the glp-1 39UTR has a temporal control that keeps translation silent until early embryogenesis and a spatial control that restricts translation to anterior cells. These controls may be mediated by distinct regulatory systems. Consistent with this, genes that affect the spatial regulation of endogenous glp-1 mRNA do not alter the timing of translation.11 Translational activation in embryos could involve inactivation of an oocyte repressor, direct promotion of translation, or both. Two possible models are that restriction of glp-1 translation to the anterior could be achieved by localizing a repressor to posterior cells, or by localizing an activator to anterior cells. One interesting aspect to studies of maternal mRNAs is their apparent instability in early embryogenesis. Many maternal mRNAs disappear rapidly in somatic cells after the four- to 24-cell stages.10 The disappearance of glp-1 mRNA occurs at least two cell cycles after localized protein synthesis is initiated and is uniform throughout most of the embryo.7,10 However, pal-1 mRNA degrades rapidly in those blastomeres that do not express PAL-1 protein, raising the possibility that regulated mRNA stability contributes to the localization of this protein.6 A similar phenomenon may occur for the mRNAs of other localized proteins such as MEX-1.12 It was argued that pal-1 mRNA instability is a secondary consequence of translational silencing, because a mutant background that at least partially restores pal-1 mRNA in anterior cells does not alter posterior localization of PAL-1 protein.6 Indeed, it has been proposed that translational control and mRNA degradation are mechanistically linked for at least some mRNAs in other organisms.13 This raises the possibility that mRNAs that appear to be localized may actually reflect local repression of translation leading to local mRNA destabilization. However, it is not known if the mech-
anisms of translational control and mRNA decay are related or distinct and their relative contributions to the localization of protein expression remain uncertain.
Trans-acting factors and pathways that control maternal mRNAs Several genes have been identified that may play roles in establishing the asymmetry of maternal mRNA translation. A somewhat oversimplified pathway of regulation in the embryo is presented in Figure 1. A system of at least six genes, the par genes, is required for the localization or expression of many factors.3 For example, par-1 is required for localization of GLP-1 expression in anterior cells,11 localization of SKN-1 in posterior cells 5 and activation of PAL-1 expression.6 Because the par genes are also required to create cell polarity and to orient cell division axes,3 they may affect mRNA translation indirectly. These genes are likely to control the distribution and activity of more specific regulators of translation. One candidate for a specific translational regulator is MEX-3, a putative KH domain RNA-binding protein.14 MEX-3 expression is first detected in developing oocytes, becomes enriched in anterior cells of embryos and is required for repression of PAL-1 translation in these cells.6,14 Moreover, MEX-3 is required to prevent PAL-1 from promoting posterior-like development in anterior cells.6 Interestingly, MEX-3 functions, at least in part, through the pal-1 39UTR since the regulation of a reporter RNA with the pal-1 39UTR is disrupted in mex-3 mutants.6 It is not known if MEX-3 binds to the pal-1 39UTR RNA elements, or acts more indirectly through other gene products. Other factors control localized gene expression in specific ways. For example, PIE-1 is required for APX-1 expression in the P2 blastomere9 and MEX-1 is required for repressing SKN-1 expression in anterior cells.5 Both PIE-1 and MEX-1 proteins contain a cysteine- rich ‘finger- like motif’ of unknown function.12,15 Conceivably, PIE-1 and MEX-1 could function as RNA-binding translational regulators. However, the developmental functions and sub-cellular locations of these factors suggests that they either act indirectly or have multiple molecular functions.12,15,16 Two general observations arise from these studies. First, different regulatory systems appear to function in the same cells. For example, both PAL-1 and 553
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SKN-1 are localized to the same posterior cells at early stages, but MEX-3 controls only PAL-1 distribution while M EX - 1 is specific for SKN - 1 localization.5,6,14 Second, virtually all of these regulators of protein localization are themselves restricted to specific blastomeres by post-transcriptional mechanisms.12,14,15 How are the different pathways coordinated, what are the regulators of these factors and where does it all begin? Interestingly, both PIE-1 and MEX- 1 proteins appear to associate with Pgranules,12,15 RNP particles that localize to the posterior of embryos beginning in the one-cell zygote.3 Perhaps P-granules serve, in part, as a macromolecular vehicle for localizing or controlling regulators of maternal mRNAs. However, spatial restriction of mRNA translation is likely to involve more than the localization of P-granules, since not all aspects of molecular asymmetry are strictly coupled to P-granule localization.3,11 Many factors that control maternal mRNA translation remain to be discovered. In the embryo, no clear candidates exist for the specific regulators of glp-1 mRNA.11 Furthermore, regulation of most maternal mRNAs begins in germ cells, yet translational repressors that function in developing oocytes have yet to be identified, with the possible exception of MEX-3 Žsee above. and GLD-1. 6,17 GLD-1 is required for several aspects of germ cell development17 and is expressed not only in the immature germ line, but is also localized to cytoplasmic particles in posterior cells of embryos between the two- and eight-cell stages.18 Because GLD-1 contains the KH-domain type of RNA-binding motif,19 it could be a regulator of several mRNAs both in germ cells and in embryos, although its role in early embyrogenesis is not yet understood. The RNA helicase-like proteins, GLH-1 and GLH-2, which are P-granule components, could also function to control maternal mRNAs in germ cells andror embryos.20 In addition, more global regulators of translation, such as those that control cytoplasmic polyadenylation of mRNAs Žsee below. or translation initiation, are likely to be required to interpret the activities of more specific mRNA regulators.
both germ line and somatic tissues. The expression of at least two sex determining genes are translationally regulated by elements in their 39UTRs. Moreover, several genes have been identified that are candidates for encoding trans-acting factors. In addition, analyses of these controls are beginning to shed light onto the underlying mechanisms. There are two sexes in C. elegans, males and hermaphrodites. Hermaphrodites are essentially female animals that first produce sperm and then switch to oogenesis. The primary signal for sex determination is the ratio of X chromosomes to sets of autosomes, such that an animal with two X chromosomes ŽXX. develops as a hermaphrodite while an animal with one X chromosome ŽXO. develops as a male Žfor an extensive review see ref. 21..
The tra-2 39UTR regulates sexual identity in the germ line and soma tra-2 encodes a large transmembrane protein that promotes female development.22,23 Male development requires that tra-2 activity is repressed. This is in part achieved by two regulatory elements, called TGEs, located in the 39UTR of tra-2 mRNA.24 Dominant gain-of-function Ž gf . mutations that remove either one or both TGEs cause enhanced tra-2 activity resulting in inappropriate feminization of both XX and XO animals; XX animals develop as females Žthey no longer make sperm. and XO animals make oocytes in the germ line and yolk in the intestine.25 These phenotypes indicate that TGEs in the tra-2 39UTR repress tra-2 activity in both germ line and somatic cells. Polysome analysis and transgenic reporter experiments indicate that the TGEs regulate tra-2 translation24 and control the length of the poly A tail.26 Moreover, TGEs contain a binding site for a factor, called DRF, that could be a regulator of translation.24 One model is that the binding of DRF to the TGEs triggers the shortening of the polyŽA. tail, which in turn leads to translational inhibition. However, it has not been demonstrated that changes in polyŽA. tail length alter tra-2 translation. DRF has not been cloned, therefore it is unclear whether DRF is composed of one or multiple factors. Genetic screens have identified two genes that contribute to both translational regulation of tra-2 and sex determination. The newly identified sex determining gene laf-1 is necessary for TGE-mediated repression.27 Similar to tra-2(gf) mutations, loss of laf-1 activity causes feminization of both XX and XO
Translational control of sexual development Recently, it has become clear that 39UTR-mediated translational controls are critical to nematode sexual development. These controls function to balance the activities that specify male and female cell fates in 554
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animals. In addition, laf-1 mutations disrupt the control of both polyŽA. tail length and translation of a reporter transgene that carries TGEs in its 39UTR.26 Perhaps laf-1 encodes a constituent of DRF. laf-1 Žand DRF., therefore, may function to maintain low levels of tra-2 activity in particular tissues at specific times of development; this control is essential to permitting spermatogenesis in hermaphrodite larvae and repression of yolk synthesis in male intestinal cells ŽFigure 2.. The function of laf-1 may extend beyond tra-2. laf-1 has a homozygous lethal phenotype that was not predicted for a loss-of-function mutation in a repressor of tra-2.27 One possible explanation is that laf-1 controls the translation of other mRNAs and the inappropriate expression of these mRNAs results in lethality. In support of this, the sex determining gene tra-1 may be controlled by TGEs ŽJan and Goodwin, unpublished results.. The tra-1 39UTR contains a TGE that is capable of repressing translation of a reporter transgene and that controls the length of the polyŽA. tail. In addition, reduction of laf-1 activity disrupts the regulation of a reporter transgene by the tra-1 TGEs. The possibility that both tra-1 and tra-2 are regulated by TGEs and laf-1 may be important in ensuring that cells adopt either a male or female state. Another sex determining gene, tra-3, appears to
promote female development in part by freeing tra-2 from translational repression.27 In contrast to wildtype animals, tra-3 mutant animals have DRF activity in embryos.27 Furthermore, reduction of tra-3 activity suppresses the ability of laf-1 mutations to disrupt regulation of the TGE-containing transgene.27 tra-3 is predicted to encode a calpain-like protease28 that is contributed maternally to embryos.29 Thus, tra-3 could act to degrade LAF-1 protein or other translational control proteins in embryos ŽFigure 2.. This function may be important to ensure female development of XX embryos. The translational control by TGEs is a conserved process that is present not only in C. elegans, but also in the nematode C. briggsae and in mammalian cells, as well.26 TGE sequences are found in the C. briggsae tra-2 gene and the human oncogene GLI. Moreover, these TGE elements can function to repress translation and gene function, both in nematodes and in mammalian cells.26 The C. briggsae tra-2 gene is homologous to the C. elegans tra-2 and is necessary for female development.30 GLI, like tra-1, is a member of the Kruppel family of transcription factors31,32 and is thought to be part of the mammalian hedgehog pathway. The similarity of translational regulation of GLI and tra-1 by TGEs may suggest that a common ancestral gene was also regulated by TGEs. These results are consistent with TGEs being an ancient mechanism for controlling gene expression that possibly regulate many other mRNAs.
The fem-3 39UTR controls the sperm-to-oocyte switch Hermaphroditism requires that a female gonad first produces sperm Ža male cell fate. and then oocytes Ža female cell fate.. 39UTR controls are important for not only the production of sperm, but also for the switch to oogenesis. As discussed above, the TGEmediated control of tra-2 translation is required for hermaphrodite spermatogenesis. The switch from spermatogenesis to oogenesis requires post-transcriptional control of the fem-3 gene through its 39UTR.33 In the germ line, fem-3 functions to promote spermatogenesis and must be down-regulated to allow oogenesis. This repression requires a 5-nt element located in the fem-3 39UTR ŽFigure 3.. Deletions or point mutations in this element cause cells that would normally develop as oocytes to be transformed into sperm.33 Northern analysis indicates that increased
Figure 2. Molecular model for how tra-3 and laf-1 modulate the translation of tra-2. ŽA. In the embryo, tra-3, a putative calpain-like protease, frees tra-2 from translational repression by removing laf-1 or DRF. ŽB. During development, tra-3 is inactive in certain tissues and at specific developmental times. Therefore, laf-1 promotes DRF binding to the TGEs and tra-2 translation is repressed resulting in male development.
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larvae.37,40,41 lin-14 encodes a broadly distributed nuclear protein that has no obvious similarity to other known proteins.42 lin-28 codes for a protein with a cold-shock domain and retroviral ŽCCHC. zinc finger motifs consistent with it acting post-transcriptionally as an RNA-binding protein.43 The mechanism by which lin-14 and lin-28 coordinate multiple cell lineages is not well understood. Critical to normal developmental timing is the temporally regulated repression of lin-14 and lin-28. Both genes are first expressed at high levels in the L1 stage, but are then repressed during the L2]L3 stages.43,44 The loss of these factors triggers the execution of successive developmental programs in the different larval stages. Both genes are regulated at the mRNA level by a conserved element, called LCE, in their 39UTRs.43 ] 45 Mutations that remove LCEs result in gain-of-function phenotypes. The lin-14 mRNA contains seven repeated LCEs while the lin-28 39UTR contains only one.43 ] 45 In wild-type animals, the lin-14 protein is present during L1s and is nearly undetectable by L2s and later stages. Gain-of-function mutations in lin-14 delete the LCEs and cause high levels of lin-14 protein to persist in later stages of development.44 However, RNase protection analysis indicates that the increase lin-14 protein does not result from changes in steady state levels of RNA,45 consistent with the LCE regulating translation. The lin-28 39UTR contains a single LCE that is capable of controlling production of lin-28 protein from a lin-28 transgene.43 Similar to lin-14, deletion of the lin-28 LCE causes high levels of lin-28 protein to continue past the L2 stage of development and results in retardation of late larval development.43 The heterochronic gene, lin-4, mediates the temporally regulated decrease in lin-14 and lin-28 protein levels. Loss-of-function mutations in lin-4 cause high levels of both lin-14 and lin-28 protein in late larval stages of development.43,44 lin-4 encodes 22-nt and 61-nt RNAs that have no open reading frames and that are complementary to the LCEs ŽFigure 4..45,46 It is proposed that the base pairing of lin-4 with the LCEs inhibits the production of protein,45,46 but how this occurs is a mystery. It is not known if lin-4 acts alone or is part of an RNP particle to regulate translation.
Figure 3. fem-3 gain-of-function mutations carry molecular lesions in a 5 nt sequence in the fem-3 39UTR. These lesions disrupt translational repression of fem-3 by its 39UTR. Shown is the fem-3 39UTR. Arrows mark single base changes. The extent of one deletion is shown below.
fem-3 activity does not likely result from increased levels of steady state mRNA, consistent with the fem-3 39UTR regulating translation.33 Like the TGEs, the fem-3 element controls the length of the polyŽA. tail.33 These results have led to the model that the fem-3 39UTR regulates translation by controlling polyŽA. tail length.33 In addition to controlling germ cell sex, fem-3 mRNA is maternally contributed to the embryo to regulate sex of the next generation.34 It is possible that the same 39UTR mechanism that represses fem-3 for the sperm-to-oocyte switch also controls maternal fem-3 mRNA. Perhaps the transient spermatogenesis evolved by using existing germ line controls of maternal mRNA. Consistent with this idea, six genes have been identified, mog-1-6 that are required for the sperm-to-oocyte switch and which are maternally required for embryogenesis.35,36 Similar to fem-3(gf) mutations, loss of mog activity results in the germ line making only sperm.35,36 The mog genes could function, either directly or indirectly, to mask fem-3 mRNA resulting in the switch to oogenesis and also to control other maternal mRNAs required for embryonic development.
Translational control of heterochronic genes and larval development The importance of translational control for C. elegans development is underscored by the recent work on the regulation of the heterochronic genes lin-14 and lin-28. Heterochronic genes govern the timing of developmental events in many different cell types.37,38,39 Loss-of-function mutations in either lin14 or lin-28 cause cell lineages of late larvae to occur prematurely in younger larvae.37 ] 39 In contrast, gainof-function mutations of these genes cause lineages typical of young larvae to be reiterated in older
Summary In C. elegans, translational control plays specific and critical roles in controlling development throughout 556
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Figure 4. Sequences in the lin-14 and lin-28 39UTRs are complementary to the lin-4 RNAs. ŽA. Predicted lin-14-lin-4 RNA duplexes in the 39UTR of lin-14 mRNA. ŽB. Predicted lin-28-lin-4 RNA duplex in the 39UTR of lin-28 mRNA.
the life of the organism. Translational control serves to coordinate the expression of various gene products at diverse places and times. Many mRNAs are transcribed in germ cells that must be differentially translated in precise patterns in the embryo. In addition, sex determination genes must be coordinately regulated in different cell lineages and at different times of development. Moreover, heterochronic genes must be controlled in diverse cell lineages of larvae. Translational regulation provides a powerful way to precisely control protein levels and distribution. Perhaps the apparent pleiotropy of some putative mRNA regulators, like GLD-1 and LAF-1, reflects the integrated regulation of multiple mRNAs. Analyses of the different 39UTR control systems in C. elegans suggests that some mechanisms may be fundamental to other eukaryotes. The TGE regulation appears to be conserved in vertebrates. The few
known suspected regulatory genes Že.g. mex-3 and gld-1. are similar to proteins in other organisms. At least some C. elegans mRNAs undergo regulated polyadenylation, a phenomenon common to many organisms.13 Moreover, non-protein coding RNAs, like lin-4, have been found in other animals, although they do not necessarily control gene expression by regulating translation. Many questions remain to be addressed. What are the trans-acting factors that control mRNA translation? Do these factors control translation directly or indirectly and what are the mechanisms? For mRNAs that are regulated in multiple cell types, are there tissue-specific or stage-specific factors? What are the pathways that control these factors during development and how are they integrated with the control of cell division and differentiation? Clearly, the established genetic, cell biological and molecular ap557
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15. Mello CC, Schubert C, Draper BW, Zhang W, Lobel B, Priess JR Ž1996. Dynamic localization of PIE-1, a maternally encoded protein required for the germ cell fate in early C. elegans embryos. Nature 382:710 16. Mello CC, Draper BW, Krause M, Weintraub H, Priess JR Ž1992. The pie-1 and mex-1 genes and maternal control of blastomere identity in early C. elegans embryos. Cell 70:163 17. Francis R, Barton MK, Kimble J, Schedl T Ž1995. gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans. Genetics 139:579 18. Jones AR, Francis R, Schedl T Ž1996. GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex- specific expression during Caenorhabditis elegans germline development. Dev Biol 180:165 19. Jones AR, Schedl T Ž1995. Mutations in gld-1, a female germ cell-specific tumor suppressor gene in Caenorhabditis elegans, affect a conserved domain also found in Src-associated protein Sam68. Gene Dev 9:1491 20. Gruidl ME, Smith PA, Kuznicki KA, McCrone JS, Kirshner J, Roussel DL, Strome S, Bennett KL Ž1996. Multiple potential germ-line helicases are components of the germ-line specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci 93:13837 21. Cline TW, Meyer BJ Ž1996. Viva la difference: males vs. females in flies vs. worms. Ann Rev Genet 30:637 22. Kuwabara PE, Okkema PG, Kimble J Ž1992. tra-2 encodes a membrane protein and may mediate cell communication in the Caenorhabditis elegans sex determination pathway. Mol Biol Cell 3:461 23. Hodgkin J Ž1980. More sex-determination mutants of Caenorhabditis elegans. Genetics 96:649 24. Goodwin EB, Okkema PG, Evans TC, Kimble J Ž1993. Translational regulation of tra-2 by its 39 untranslated region controls sexual identity in C. elegans. Cell 75:329 25. Doniach T Ž1986. Activity of the sex-determining gene tra-2 is modulated to allow spermatogenesis in the C. elegans hermaphrodite. Genetics 114:53 26. Jan E, Yoon JW, Walterhouse D, Iannaccone P, Goodwin EB Ž1997. Conservation of the C. elegans tra-2 39UTR translational control. EMBO J Žin press. 27. Goodwin EB, Hofstra K, Hurney CA, Mango S, Kimble J Ž1997. A genetic pathway for regulation of tra-2 translation. Development 124:749 28. Barnes TM, Hodgkin J Ž1996. The tra-3 sex determination gene of Caenorhabditis elegans encodes a member of the calpain regulatory protease family. EMBO J 15:4477 29. Hodgkin J, Brenner S Ž1977. Mutations causing transformation of sexual phenotype in the nematode Caenorhabditis elegans. Genetics 86:275 30. Kuwabara PE Ž1996. Interspecies comparison reveals evolution of control regions in the nematode sex-determining gene tra-2. Genetics 144:597 31. Kinzler KW, Bigner SH, Bigner DD, Trent JM, Law ML, O’Brien SJ, Wong AJ, Vogelstein B Ž1987. Identification of an amplified, highly expressed gene in a human glioma. Science 236:70 32. Ruppert JM, Kinzler KW, Wong AJ, Bigner SH, Kao FT, Law ML, Seuanez HN, O’Brien SJ, Vogelstein B Ž1988. The GLIKruppel family of human genes. Mol Cell Biol 8:3104 33. Ahringer J, Kimble J Ž1991. Control of the sperm]oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 39 untranslated region. Nature 349:346 34. Ahringer J, Rosenquist TA, Lawson DN, Kimble J Ž1992. The Caenorhabditis elegans sex determining gene fem-3 is regulated post-transcriptionally. EMBO J 11:2303 35. Graham PL, Kimble J Ž1993. The mog-1 gene is required for the switch from spermatogenesis to oogenesis in Caenorhabditis elegans. Genetics 133:919
proaches in C. elegans has much to offer in addressing these questions. However, the continued development of biochemical methods will also be critical to future research.
Acknowledgements We are grateful to Victor Ambrose, Maria Gallegos, Judith Kimble, Craig Mello, Eric Moss and Phil Olsen for discussing unpublished results and clarifying information. We thank Ken Geles, Laura Graves and Eric Jan for critical reading of the manuscript. This work was supported in part by grants from the NIH and the Council for Tobacco Research to E.B.G. and from the American Cancer Society to the Univ of Col. Cancer Center to T.C.E.
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36. Graham PL, Schedl T, Kimble J Ž1993. More mog genes that influence the switch from spermatogenesis to oogenesis in the hermaphrodite germ line of Caenorhabditis elegans. Dev Genet 14:471 37. Ambros V, Horvitz HR Ž1984. Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226:409 38. Ambrose V Ž1989. A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans. Cell 57:49 39. Euling S, Ambrose V Ž1996. Heterochronic genes control cell cycle progress and developmental competence of C. elegans vulva precursor cells. Cell 84:667 40. Chalfie M, Horvitz HR, Dulston, JE Ž1981. Mutations that lead to reiterations in the cell lineages of C. elegans. Cell 24:59 41. Ambrose V, Horvitz HR Ž1987.. The lin-14 locus of Caenorhabditis elegans controls the time of expression of specific postembryonic developmental events. Gene Dev 1:398
42. Ruvkin G, Giusto J Ž1989. The Caenorhabditis elegans heterochronic gene lin-14 encodes a nuclear protein that forms a temporal developmental switch. Nature 338:313 43. Moss EG, Lee RC, Ambrose V Ž1997. The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-14 RNA. Cell 88:637 44. Wightman B, Burglin TR, Gatto, J, Arasu P, Ruvkin G Ž1991. Negative regulatory sequences in the lin-14 39-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development. Gene Dev 5:1813 45. Wightman B, Ha I, Ruvkun G Ž1993. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75:855 46. Lee RC, Feinbaum RL, Ambrose V Ž1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843
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Translational control of development in C. elegans Elizabeth B. GoodwinU and Thomas C. Evans†
Translational control by the 39untranslated regions (39UTRs) of mRNAs contributes to important events throughout the development of C. elegans. In oocytes and early embryos, maternal mRNAs are controlled by 39UTR elements to restrict translation of their protein products to specific blastomeres. Localized translation is probably critical for specifying blastomere identity. In both germline and somatic cells, mRNAs from sex determining genes are translationally repressed by 39UTR controls. These controls balance the activities that specify male and female cell fates. During larval development, the temporal sequence of cell lineages requires 39UTR-mediated regulation of heterochronic genes by a small non-protein coding RNA. We review what is known about these translational control mechanisms in C. elegans. This overview illustrates that translational control by 39UTR elements is a powerful mechanism for regulating the expression of multiple gene products in diverse cell types during development of a multi-cellular animal.
over, it is largely unknown how these mechanisms are connected to other pathways Že.g. signal transduction and cell cycle controls. to regulate developmental decisions. In this review, we discuss the role of translational control in the development of the nematode C. elegans. Work on C. elegans has revealed that 39UTRmediated regulation governs a wide range of developmental events throughout the animal’s life. So far, translational regulation has been implicated in the control of early embryogenesis, sex determination of both germ line and somatic cells and the temporal execution of cell lineages during larval development. Using the genetic, cell biological and molecular approaches available in the worm, cis-acting elements and trans-acting factors that control mRNA translation are being identified and pathways that integrate these factors with development are being discovered. For this discussion, we define translational control somewhat broadly; regulation of translation may occur by a variety of different mechamisms. In most examples of translationally controlled mRNAs in C. elegans, it is not known if 39UTRs control ribosome initiation or elongation. 39UTR elements and transacting factors could directly interact with the translational apparatus, or they could sequester mRNAs away from translation components. Furthermore, in some cases regulation of mRNA transport from the nucleus has not been ruled. Future analysis of the cellular and molecular details will certainly be of interest in resolving these issues.
Key words: translational control r 39UTR r C. elegans r sex determination r heterochronic genes Q1997 Academic Press Ltd
DURING ANIMAL DEVELOPMENT, proteins that govern cell fate are expressed at defined levels, at exact times and in specific cells. This precise control of gene expression is essential to specify when cells divide, where they go and what they become. In both vertebrates and invertebrates, recent work indicates that the control of translation by elements in the 39untranslated region Ž39UTR. of mRNAs is critical to regulating a number of developmental processes Žsee refs. 1 and 2 for reviews.. Yet, the mechanisms of 39UTR-mediated regulation remain obscure. More-
Maternal mRNA regulation and development of the early embryo In C. elegans, as in most animal species, the early development of embryos depends on mRNAs and proteins provided by oocytes. These maternal gene products are transcribed in germ cells and thus must be controlled post-transcriptionally in embryos. Recent work demonstrates that precise regulation of maternal factors is critical to creating distinct identi-
U
From the Department of Cell and Molecular Biology and the Lurie Cancer Center, Northwestern University Medical School, 303 East Chicago Ave., Chicago IL 60611 USA and †Department of Cell and Structural Biology, University of Colorado Health Science Center, Denver, CO 80262 USA Q1997 Academic Press Ltd 1084-9521r 97r 060551q 09 $25.00r 0r sr970180
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ceiving and transducing signals from posterior cells.4 One putative GLP-1 ligand, the APX-1 protein, is expressed exclusively in the posterior-most cell of the four-cell embryo, called P2.9 The P2 localization of the APX-1 ligand and the anterior localization of the GLP-1 receptor is probably needed to spatially constrain cell signaling so that the two anterior blastomeres can adopt different cell fates.4 In addition to the localization of these factors, the production of all of these proteins are temporally regulated; proteins are generally not present in oocytes, they begin to appear between the one- and four-cell stages and most factors disappear by the eight- to 24-cell stages. Therefore early C. elegans embryogenesis relies on elaborate temporal and spatial control systems that restrict key factors to specific cells in the embryo. What are the mechanisms that mediate this remarkable regulation? The glp-1, skn-1, apx-1 and pal-1 mRNAs are transcribed in germ cells and are, at least initially, evenly distributed in the cytoplasms of oocytes and all cells of the early embryo.6,7,9,10 Therefore the unique locations of these proteins appear to result from distinct patterns of localized mRNA translation andror protein stability. Reporter RNA experiments showed that the glp-1 39UTR was sufficient to restrict the translation of a heterologous RNA to anterior cells of early embryos, without affecting lo-
ties for the early blastomeres of the embryo. Several observations suggest that the control of maternal mRNA translation plays a prominent role in this process. The first cell division in the embryo is asymmetric, generating an anterior blastomere and a posterior blastomere which differ in composition and developmental potential Žreviewed in refs. 3 and 4..3,4 With each subsequent cell division, each daughter blastomere aquires a unique identity either by inheriting a particular set of factors following asymmetric cell division, or through specific interactions with neighboring cells. Mutant screens have identified several genes whose maternally contributed gene products are critical to the specification of early cell identities. The precise pattern of early embryogensis requires the localization of the protein products of some of these genes to specific blastomeres ŽFigure 1.. By the four-cell stage, the two putative transcription factors SKN-1 and PAL-1 are only present in the posterior cells.5,6 These two proteins appear to function as determinants of posterior cell types;5,6,8 their localization to posterior cells may be critical to restricting posterior fates to these cells.5,6 In contrast, the membrane-bound receptor GLP-1 is expressed only in the two anterior blastomeres at the four-cell stage.7 GLP-1 regulates the determination of anterior cells by re-
Figure 1. A pathway for the localization of maternal factors during early C. elegans embryogenesis. Polarity is created in the 1 cell zygote leading to the posterior localization of P-granules Žsmall dots. and other asymmetries. Anterior Žshaded. and posterior Žunshaded. cells arise following cell division. The membrane proteins GLP-1 Žblack. and APX-1 Žstippled., and the nuclear proteins SKN-1 Žgray. and PAL-1 Žblack. are localized by the four cell stages. The embryo at the far right represents the different identieis for all cells arising from the localization of maternal factors and a cell interaction Žarrow.. Known factors Že.g. PAR proteins. and predicted unknown factors Ž? . that control different aspects of polarity and maternal mRNA regulation are indicated. Some factors are required to promote protein expression Že.g. PIE-1 . while others repress expression in non-expressing cells Že.g. MEX-1 and MEX-3 ..
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calization of the RNA.7 A similar experiment showed that the pal-1 39UTR largely restricts reporter protein expression to posterior cells.6 These results suggest that translational controls via the 39UTRs of glp-1 and pal-1 at least in part create the asymmetric distribution of their protein products. Analyses of how the translation of maternal mRNAs is controlled have only just begun. As described above, the glp-1 39UTR confers regulation on a reporter RNA; translation is repressed in oocytes, is activated in early embryos and is localized to anterior cells.7 A U-rich region at the 39 end is required to repress translation in ooctyes. In contrast, a distinct region is required for repressing translation in posterior cells, but is not required for oocyte repression. Therefore the glp-1 39UTR has a temporal control that keeps translation silent until early embryogenesis and a spatial control that restricts translation to anterior cells. These controls may be mediated by distinct regulatory systems. Consistent with this, genes that affect the spatial regulation of endogenous glp-1 mRNA do not alter the timing of translation.11 Translational activation in embryos could involve inactivation of an oocyte repressor, direct promotion of translation, or both. Two possible models are that restriction of glp-1 translation to the anterior could be achieved by localizing a repressor to posterior cells, or by localizing an activator to anterior cells. One interesting aspect to studies of maternal mRNAs is their apparent instability in early embryogenesis. Many maternal mRNAs disappear rapidly in somatic cells after the four- to 24-cell stages.10 The disappearance of glp-1 mRNA occurs at least two cell cycles after localized protein synthesis is initiated and is uniform throughout most of the embryo.7,10 However, pal-1 mRNA degrades rapidly in those blastomeres that do not express PAL-1 protein, raising the possibility that regulated mRNA stability contributes to the localization of this protein.6 A similar phenomenon may occur for the mRNAs of other localized proteins such as MEX-1.12 It was argued that pal-1 mRNA instability is a secondary consequence of translational silencing, because a mutant background that at least partially restores pal-1 mRNA in anterior cells does not alter posterior localization of PAL-1 protein.6 Indeed, it has been proposed that translational control and mRNA degradation are mechanistically linked for at least some mRNAs in other organisms.13 This raises the possibility that mRNAs that appear to be localized may actually reflect local repression of translation leading to local mRNA destabilization. However, it is not known if the mech-
anisms of translational control and mRNA decay are related or distinct and their relative contributions to the localization of protein expression remain uncertain.
Trans-acting factors and pathways that control maternal mRNAs Several genes have been identified that may play roles in establishing the asymmetry of maternal mRNA translation. A somewhat oversimplified pathway of regulation in the embryo is presented in Figure 1. A system of at least six genes, the par genes, is required for the localization or expression of many factors.3 For example, par-1 is required for localization of GLP-1 expression in anterior cells,11 localization of SKN-1 in posterior cells 5 and activation of PAL-1 expression.6 Because the par genes are also required to create cell polarity and to orient cell division axes,3 they may affect mRNA translation indirectly. These genes are likely to control the distribution and activity of more specific regulators of translation. One candidate for a specific translational regulator is MEX-3, a putative KH domain RNA-binding protein.14 MEX-3 expression is first detected in developing oocytes, becomes enriched in anterior cells of embryos and is required for repression of PAL-1 translation in these cells.6,14 Moreover, MEX-3 is required to prevent PAL-1 from promoting posterior-like development in anterior cells.6 Interestingly, MEX-3 functions, at least in part, through the pal-1 39UTR since the regulation of a reporter RNA with the pal-1 39UTR is disrupted in mex-3 mutants.6 It is not known if MEX-3 binds to the pal-1 39UTR RNA elements, or acts more indirectly through other gene products. Other factors control localized gene expression in specific ways. For example, PIE-1 is required for APX-1 expression in the P2 blastomere9 and MEX-1 is required for repressing SKN-1 expression in anterior cells.5 Both PIE-1 and MEX-1 proteins contain a cysteine- rich ‘finger- like motif’ of unknown function.12,15 Conceivably, PIE-1 and MEX-1 could function as RNA-binding translational regulators. However, the developmental functions and sub-cellular locations of these factors suggests that they either act indirectly or have multiple molecular functions.12,15,16 Two general observations arise from these studies. First, different regulatory systems appear to function in the same cells. For example, both PAL-1 and 553
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SKN-1 are localized to the same posterior cells at early stages, but MEX-3 controls only PAL-1 distribution while M EX - 1 is specific for SKN - 1 localization.5,6,14 Second, virtually all of these regulators of protein localization are themselves restricted to specific blastomeres by post-transcriptional mechanisms.12,14,15 How are the different pathways coordinated, what are the regulators of these factors and where does it all begin? Interestingly, both PIE-1 and MEX- 1 proteins appear to associate with Pgranules,12,15 RNP particles that localize to the posterior of embryos beginning in the one-cell zygote.3 Perhaps P-granules serve, in part, as a macromolecular vehicle for localizing or controlling regulators of maternal mRNAs. However, spatial restriction of mRNA translation is likely to involve more than the localization of P-granules, since not all aspects of molecular asymmetry are strictly coupled to P-granule localization.3,11 Many factors that control maternal mRNA translation remain to be discovered. In the embryo, no clear candidates exist for the specific regulators of glp-1 mRNA.11 Furthermore, regulation of most maternal mRNAs begins in germ cells, yet translational repressors that function in developing oocytes have yet to be identified, with the possible exception of MEX-3 Žsee above. and GLD-1. 6,17 GLD-1 is required for several aspects of germ cell development17 and is expressed not only in the immature germ line, but is also localized to cytoplasmic particles in posterior cells of embryos between the two- and eight-cell stages.18 Because GLD-1 contains the KH-domain type of RNA-binding motif,19 it could be a regulator of several mRNAs both in germ cells and in embryos, although its role in early embyrogenesis is not yet understood. The RNA helicase-like proteins, GLH-1 and GLH-2, which are P-granule components, could also function to control maternal mRNAs in germ cells andror embryos.20 In addition, more global regulators of translation, such as those that control cytoplasmic polyadenylation of mRNAs Žsee below. or translation initiation, are likely to be required to interpret the activities of more specific mRNA regulators.
both germ line and somatic tissues. The expression of at least two sex determining genes are translationally regulated by elements in their 39UTRs. Moreover, several genes have been identified that are candidates for encoding trans-acting factors. In addition, analyses of these controls are beginning to shed light onto the underlying mechanisms. There are two sexes in C. elegans, males and hermaphrodites. Hermaphrodites are essentially female animals that first produce sperm and then switch to oogenesis. The primary signal for sex determination is the ratio of X chromosomes to sets of autosomes, such that an animal with two X chromosomes ŽXX. develops as a hermaphrodite while an animal with one X chromosome ŽXO. develops as a male Žfor an extensive review see ref. 21..
The tra-2 39UTR regulates sexual identity in the germ line and soma tra-2 encodes a large transmembrane protein that promotes female development.22,23 Male development requires that tra-2 activity is repressed. This is in part achieved by two regulatory elements, called TGEs, located in the 39UTR of tra-2 mRNA.24 Dominant gain-of-function Ž gf . mutations that remove either one or both TGEs cause enhanced tra-2 activity resulting in inappropriate feminization of both XX and XO animals; XX animals develop as females Žthey no longer make sperm. and XO animals make oocytes in the germ line and yolk in the intestine.25 These phenotypes indicate that TGEs in the tra-2 39UTR repress tra-2 activity in both germ line and somatic cells. Polysome analysis and transgenic reporter experiments indicate that the TGEs regulate tra-2 translation24 and control the length of the poly A tail.26 Moreover, TGEs contain a binding site for a factor, called DRF, that could be a regulator of translation.24 One model is that the binding of DRF to the TGEs triggers the shortening of the polyŽA. tail, which in turn leads to translational inhibition. However, it has not been demonstrated that changes in polyŽA. tail length alter tra-2 translation. DRF has not been cloned, therefore it is unclear whether DRF is composed of one or multiple factors. Genetic screens have identified two genes that contribute to both translational regulation of tra-2 and sex determination. The newly identified sex determining gene laf-1 is necessary for TGE-mediated repression.27 Similar to tra-2(gf) mutations, loss of laf-1 activity causes feminization of both XX and XO
Translational control of sexual development Recently, it has become clear that 39UTR-mediated translational controls are critical to nematode sexual development. These controls function to balance the activities that specify male and female cell fates in 554
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animals. In addition, laf-1 mutations disrupt the control of both polyŽA. tail length and translation of a reporter transgene that carries TGEs in its 39UTR.26 Perhaps laf-1 encodes a constituent of DRF. laf-1 Žand DRF., therefore, may function to maintain low levels of tra-2 activity in particular tissues at specific times of development; this control is essential to permitting spermatogenesis in hermaphrodite larvae and repression of yolk synthesis in male intestinal cells ŽFigure 2.. The function of laf-1 may extend beyond tra-2. laf-1 has a homozygous lethal phenotype that was not predicted for a loss-of-function mutation in a repressor of tra-2.27 One possible explanation is that laf-1 controls the translation of other mRNAs and the inappropriate expression of these mRNAs results in lethality. In support of this, the sex determining gene tra-1 may be controlled by TGEs ŽJan and Goodwin, unpublished results.. The tra-1 39UTR contains a TGE that is capable of repressing translation of a reporter transgene and that controls the length of the polyŽA. tail. In addition, reduction of laf-1 activity disrupts the regulation of a reporter transgene by the tra-1 TGEs. The possibility that both tra-1 and tra-2 are regulated by TGEs and laf-1 may be important in ensuring that cells adopt either a male or female state. Another sex determining gene, tra-3, appears to
promote female development in part by freeing tra-2 from translational repression.27 In contrast to wildtype animals, tra-3 mutant animals have DRF activity in embryos.27 Furthermore, reduction of tra-3 activity suppresses the ability of laf-1 mutations to disrupt regulation of the TGE-containing transgene.27 tra-3 is predicted to encode a calpain-like protease28 that is contributed maternally to embryos.29 Thus, tra-3 could act to degrade LAF-1 protein or other translational control proteins in embryos ŽFigure 2.. This function may be important to ensure female development of XX embryos. The translational control by TGEs is a conserved process that is present not only in C. elegans, but also in the nematode C. briggsae and in mammalian cells, as well.26 TGE sequences are found in the C. briggsae tra-2 gene and the human oncogene GLI. Moreover, these TGE elements can function to repress translation and gene function, both in nematodes and in mammalian cells.26 The C. briggsae tra-2 gene is homologous to the C. elegans tra-2 and is necessary for female development.30 GLI, like tra-1, is a member of the Kruppel family of transcription factors31,32 and is thought to be part of the mammalian hedgehog pathway. The similarity of translational regulation of GLI and tra-1 by TGEs may suggest that a common ancestral gene was also regulated by TGEs. These results are consistent with TGEs being an ancient mechanism for controlling gene expression that possibly regulate many other mRNAs.
The fem-3 39UTR controls the sperm-to-oocyte switch Hermaphroditism requires that a female gonad first produces sperm Ža male cell fate. and then oocytes Ža female cell fate.. 39UTR controls are important for not only the production of sperm, but also for the switch to oogenesis. As discussed above, the TGEmediated control of tra-2 translation is required for hermaphrodite spermatogenesis. The switch from spermatogenesis to oogenesis requires post-transcriptional control of the fem-3 gene through its 39UTR.33 In the germ line, fem-3 functions to promote spermatogenesis and must be down-regulated to allow oogenesis. This repression requires a 5-nt element located in the fem-3 39UTR ŽFigure 3.. Deletions or point mutations in this element cause cells that would normally develop as oocytes to be transformed into sperm.33 Northern analysis indicates that increased
Figure 2. Molecular model for how tra-3 and laf-1 modulate the translation of tra-2. ŽA. In the embryo, tra-3, a putative calpain-like protease, frees tra-2 from translational repression by removing laf-1 or DRF. ŽB. During development, tra-3 is inactive in certain tissues and at specific developmental times. Therefore, laf-1 promotes DRF binding to the TGEs and tra-2 translation is repressed resulting in male development.
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larvae.37,40,41 lin-14 encodes a broadly distributed nuclear protein that has no obvious similarity to other known proteins.42 lin-28 codes for a protein with a cold-shock domain and retroviral ŽCCHC. zinc finger motifs consistent with it acting post-transcriptionally as an RNA-binding protein.43 The mechanism by which lin-14 and lin-28 coordinate multiple cell lineages is not well understood. Critical to normal developmental timing is the temporally regulated repression of lin-14 and lin-28. Both genes are first expressed at high levels in the L1 stage, but are then repressed during the L2]L3 stages.43,44 The loss of these factors triggers the execution of successive developmental programs in the different larval stages. Both genes are regulated at the mRNA level by a conserved element, called LCE, in their 39UTRs.43 ] 45 Mutations that remove LCEs result in gain-of-function phenotypes. The lin-14 mRNA contains seven repeated LCEs while the lin-28 39UTR contains only one.43 ] 45 In wild-type animals, the lin-14 protein is present during L1s and is nearly undetectable by L2s and later stages. Gain-of-function mutations in lin-14 delete the LCEs and cause high levels of lin-14 protein to persist in later stages of development.44 However, RNase protection analysis indicates that the increase lin-14 protein does not result from changes in steady state levels of RNA,45 consistent with the LCE regulating translation. The lin-28 39UTR contains a single LCE that is capable of controlling production of lin-28 protein from a lin-28 transgene.43 Similar to lin-14, deletion of the lin-28 LCE causes high levels of lin-28 protein to continue past the L2 stage of development and results in retardation of late larval development.43 The heterochronic gene, lin-4, mediates the temporally regulated decrease in lin-14 and lin-28 protein levels. Loss-of-function mutations in lin-4 cause high levels of both lin-14 and lin-28 protein in late larval stages of development.43,44 lin-4 encodes 22-nt and 61-nt RNAs that have no open reading frames and that are complementary to the LCEs ŽFigure 4..45,46 It is proposed that the base pairing of lin-4 with the LCEs inhibits the production of protein,45,46 but how this occurs is a mystery. It is not known if lin-4 acts alone or is part of an RNP particle to regulate translation.
Figure 3. fem-3 gain-of-function mutations carry molecular lesions in a 5 nt sequence in the fem-3 39UTR. These lesions disrupt translational repression of fem-3 by its 39UTR. Shown is the fem-3 39UTR. Arrows mark single base changes. The extent of one deletion is shown below.
fem-3 activity does not likely result from increased levels of steady state mRNA, consistent with the fem-3 39UTR regulating translation.33 Like the TGEs, the fem-3 element controls the length of the polyŽA. tail.33 These results have led to the model that the fem-3 39UTR regulates translation by controlling polyŽA. tail length.33 In addition to controlling germ cell sex, fem-3 mRNA is maternally contributed to the embryo to regulate sex of the next generation.34 It is possible that the same 39UTR mechanism that represses fem-3 for the sperm-to-oocyte switch also controls maternal fem-3 mRNA. Perhaps the transient spermatogenesis evolved by using existing germ line controls of maternal mRNA. Consistent with this idea, six genes have been identified, mog-1-6 that are required for the sperm-to-oocyte switch and which are maternally required for embryogenesis.35,36 Similar to fem-3(gf) mutations, loss of mog activity results in the germ line making only sperm.35,36 The mog genes could function, either directly or indirectly, to mask fem-3 mRNA resulting in the switch to oogenesis and also to control other maternal mRNAs required for embryonic development.
Translational control of heterochronic genes and larval development The importance of translational control for C. elegans development is underscored by the recent work on the regulation of the heterochronic genes lin-14 and lin-28. Heterochronic genes govern the timing of developmental events in many different cell types.37,38,39 Loss-of-function mutations in either lin14 or lin-28 cause cell lineages of late larvae to occur prematurely in younger larvae.37 ] 39 In contrast, gainof-function mutations of these genes cause lineages typical of young larvae to be reiterated in older
Summary In C. elegans, translational control plays specific and critical roles in controlling development throughout 556
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Figure 4. Sequences in the lin-14 and lin-28 39UTRs are complementary to the lin-4 RNAs. ŽA. Predicted lin-14-lin-4 RNA duplexes in the 39UTR of lin-14 mRNA. ŽB. Predicted lin-28-lin-4 RNA duplex in the 39UTR of lin-28 mRNA.
the life of the organism. Translational control serves to coordinate the expression of various gene products at diverse places and times. Many mRNAs are transcribed in germ cells that must be differentially translated in precise patterns in the embryo. In addition, sex determination genes must be coordinately regulated in different cell lineages and at different times of development. Moreover, heterochronic genes must be controlled in diverse cell lineages of larvae. Translational regulation provides a powerful way to precisely control protein levels and distribution. Perhaps the apparent pleiotropy of some putative mRNA regulators, like GLD-1 and LAF-1, reflects the integrated regulation of multiple mRNAs. Analyses of the different 39UTR control systems in C. elegans suggests that some mechanisms may be fundamental to other eukaryotes. The TGE regulation appears to be conserved in vertebrates. The few
known suspected regulatory genes Že.g. mex-3 and gld-1. are similar to proteins in other organisms. At least some C. elegans mRNAs undergo regulated polyadenylation, a phenomenon common to many organisms.13 Moreover, non-protein coding RNAs, like lin-4, have been found in other animals, although they do not necessarily control gene expression by regulating translation. Many questions remain to be addressed. What are the trans-acting factors that control mRNA translation? Do these factors control translation directly or indirectly and what are the mechanisms? For mRNAs that are regulated in multiple cell types, are there tissue-specific or stage-specific factors? What are the pathways that control these factors during development and how are they integrated with the control of cell division and differentiation? Clearly, the established genetic, cell biological and molecular ap557
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15. Mello CC, Schubert C, Draper BW, Zhang W, Lobel B, Priess JR Ž1996. Dynamic localization of PIE-1, a maternally encoded protein required for the germ cell fate in early C. elegans embryos. Nature 382:710 16. Mello CC, Draper BW, Krause M, Weintraub H, Priess JR Ž1992. The pie-1 and mex-1 genes and maternal control of blastomere identity in early C. elegans embryos. Cell 70:163 17. Francis R, Barton MK, Kimble J, Schedl T Ž1995. gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans. Genetics 139:579 18. Jones AR, Francis R, Schedl T Ž1996. GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex- specific expression during Caenorhabditis elegans germline development. Dev Biol 180:165 19. Jones AR, Schedl T Ž1995. Mutations in gld-1, a female germ cell-specific tumor suppressor gene in Caenorhabditis elegans, affect a conserved domain also found in Src-associated protein Sam68. Gene Dev 9:1491 20. Gruidl ME, Smith PA, Kuznicki KA, McCrone JS, Kirshner J, Roussel DL, Strome S, Bennett KL Ž1996. Multiple potential germ-line helicases are components of the germ-line specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci 93:13837 21. Cline TW, Meyer BJ Ž1996. Viva la difference: males vs. females in flies vs. worms. Ann Rev Genet 30:637 22. Kuwabara PE, Okkema PG, Kimble J Ž1992. tra-2 encodes a membrane protein and may mediate cell communication in the Caenorhabditis elegans sex determination pathway. Mol Biol Cell 3:461 23. Hodgkin J Ž1980. More sex-determination mutants of Caenorhabditis elegans. Genetics 96:649 24. Goodwin EB, Okkema PG, Evans TC, Kimble J Ž1993. Translational regulation of tra-2 by its 39 untranslated region controls sexual identity in C. elegans. Cell 75:329 25. Doniach T Ž1986. Activity of the sex-determining gene tra-2 is modulated to allow spermatogenesis in the C. elegans hermaphrodite. Genetics 114:53 26. Jan E, Yoon JW, Walterhouse D, Iannaccone P, Goodwin EB Ž1997. Conservation of the C. elegans tra-2 39UTR translational control. EMBO J Žin press. 27. Goodwin EB, Hofstra K, Hurney CA, Mango S, Kimble J Ž1997. A genetic pathway for regulation of tra-2 translation. Development 124:749 28. Barnes TM, Hodgkin J Ž1996. The tra-3 sex determination gene of Caenorhabditis elegans encodes a member of the calpain regulatory protease family. EMBO J 15:4477 29. Hodgkin J, Brenner S Ž1977. Mutations causing transformation of sexual phenotype in the nematode Caenorhabditis elegans. Genetics 86:275 30. Kuwabara PE Ž1996. Interspecies comparison reveals evolution of control regions in the nematode sex-determining gene tra-2. Genetics 144:597 31. Kinzler KW, Bigner SH, Bigner DD, Trent JM, Law ML, O’Brien SJ, Wong AJ, Vogelstein B Ž1987. Identification of an amplified, highly expressed gene in a human glioma. Science 236:70 32. Ruppert JM, Kinzler KW, Wong AJ, Bigner SH, Kao FT, Law ML, Seuanez HN, O’Brien SJ, Vogelstein B Ž1988. The GLIKruppel family of human genes. Mol Cell Biol 8:3104 33. Ahringer J, Kimble J Ž1991. Control of the sperm]oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 39 untranslated region. Nature 349:346 34. Ahringer J, Rosenquist TA, Lawson DN, Kimble J Ž1992. The Caenorhabditis elegans sex determining gene fem-3 is regulated post-transcriptionally. EMBO J 11:2303 35. Graham PL, Kimble J Ž1993. The mog-1 gene is required for the switch from spermatogenesis to oogenesis in Caenorhabditis elegans. Genetics 133:919
proaches in C. elegans has much to offer in addressing these questions. However, the continued development of biochemical methods will also be critical to future research.
Acknowledgements We are grateful to Victor Ambrose, Maria Gallegos, Judith Kimble, Craig Mello, Eric Moss and Phil Olsen for discussing unpublished results and clarifying information. We thank Ken Geles, Laura Graves and Eric Jan for critical reading of the manuscript. This work was supported in part by grants from the NIH and the Council for Tobacco Research to E.B.G. and from the American Cancer Society to the Univ of Col. Cancer Center to T.C.E.
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