- Email: [email protected]
Translational repressors in Drosophila
572
Review
TRENDS in Genetics Vol.18 No.11 November 2002
Translational repressors in Drosophila Kellie A. Dean, Aneel K. Aggarwal and Robin P. Wharton Translational regulation is an important aspect of gene regulation, particularly during early development of the fruit fly embryo when transcriptional mechanisms are untenable. Study of pattern formation and dosage compensation has identified several repressors that bind discrete sites in the untranslated portions of target mRNAs. These repressors do not work in isolation – each binds multiple sites in the appropriate mRNA, and the resulting RNA–protein complexes appear to recruit co-repressors by a variety of mechanisms.
which regulate translation of caudal and tramtrack69 mRNAs, respectively [3,4]). Most of the translation factors that pass muster are involved in early development, although the first to be discussed below, Sex-lethal (Sxl), is required for dosage compensation and sex determination throughout the life of the fly. Sxl: a regulator of both splicing and translation
Published online: 24 September 2002
Kellie A. Dean Robin P. Wharton* Howard Hughes Medical Institute, Dept of Molecular Genetics and Microbiology, Box 3657, Duke University Medical Center, Durham, NC 27710, USA. *e-mail: [email protected] Aneel K. Aggarwal Structural Biology Program, Dept of Physiology and Biophysics, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA.
Translational regulation of mRNA is a widespread strategy that is used to govern fundamental cellular processes (e.g. iron homeostasis in mammals), as well as esoteric events such as the sperm–oocyte switch in the hermaphroditic nematode Caenorhabditis elegans (reviewed in [1,2]). During stages of the life cycle when the genome is transcriptionally inaccessible, translational control (and other post-transcriptional mechanisms) have predominant roles; for example, during the DNA compaction of spermatogenesis or in anucleate erythrocytes. Establishment of the body plan in the Drosophila embryo takes place during one such period of transcriptional reticence. The embryonic pattern derives from localized maternal products that are deposited in the oocyte, which itself is arrested in meiosis and transcriptionally inert. Subsequent patterning is elaborated during the first few hours of embryonic development when the nuclei are in manic, 10-min cleavage cycles that are incompatible with extensive transcription. As a consequence of these restrictions, translational mechanisms have a significant role in early pattern formation. Progress in the field has been led by study of inhibitors; in the past few years, the early embryo has yielded six novel translational repressors and two novel RNA-binding motifs. This review is not intended to be comprehensive, even among Drosophila proteins. Rather, it is limited to a discussion of translational repressors defined by three criteria. First, in addition to discordant accumulation of mRNA and protein in two different cell types (or subcellular compartments), repression is conferred by sequences in the untranslated regions of the mRNA. Second, one or more factors bind to discrete sites in the mRNA. And third, mutations in the gene encoding the binding factor have the same biological and molecular consequences as mutations in its binding site(s) within the mRNA. These criteria avoid the possibility that apparent translational control is in fact the result of differential protein degradation. They also narrowly exclude some interesting factors (such as Bicoid and Musashi, http://tig.trends.com
Sxl is a master regulator, required in females and obligatorily absent in males. It was originally shown to be a splicing factor during sex determination, governing the production of sex-specific mRNA isoforms at the head of a cascade of regulated splicing events. In this guise, Sxl recognizes uridylate-rich sequences near splicing sites using a pair of RRMs (RNA-recognition motifs) [5,6]. Sxl also controls dosage compensation, and it was natural to assume it would do so by similar methods. However, two groups have shown independently that Sxl has a more interesting role than initially suspected. One of the critical effectors of dosage compensation is Msl-2 protein, which accumulates in males but not in females although both contain its mRNA [7–9]. However, the structure of the mRNA is different in each sex: the female mRNA isoform bears sequences in the 5′ untranslated region (UTR) that are excised as an intron to generate the male isoform. Within this intron are two Sxl-binding sites, and Sxl activity is required to block excision of the intron in females (Fig. 1). The key experiment indicting Sxl as a translational repressor was mutation of the splice acceptor and donor sites that define the intron. Bashaw and Baker [10] drove expression of a suitably modified msl-2 gene with a strong constitutive promoter in both cultured S2 cells and transgenic flies; Kuroda and colleagues pursued the more prudent course of modifying an otherwise wild type msl-2 transgene (thereby relying on endogenous transcriptional and processing signals to generate normal levels of mRNA precursor) [11]. Happily, both groups reached the same conclusion: even if the intron is forcibly included, binding of Sxl represses translation of the mRNA. Thus, Sxl prevents excision of its binding sites in the 5′ UTR so that it can subsequently repress translation from the selfsame sites (in females). Further work uncovered a complexity shared by all of the repressors described in this review – full repression in vivo requires occupancy of multiple binding sites in the mRNA. In the case of Sxl and msl-2 mRNA, one of the binding sites in the 5′ UTR mediates most of the repression, but the nearby site, as well as a cluster of sites near the 3′ terminus of the mRNA, contribute significantly.
0168-9525/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(02)02792-0
Review
TRENDS in Genetics Vol.18 No.11 November 2002
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nos
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Fig. 1. Regulation of msl-2 mRNA by Sxl. In females, Sxl binds to sites in the 5′ UTR of the pre-mRNA, preventing their excision so that cytoplasmic Sxl can subsequently repress translation. In males, the 5′ intron is excised in the absence of Sxl and the mRNA is not repressed.
PUM BRAT SMG
Gebauer and colleagues have begun to explore the mechanism of Sxl-dependent repression using an in vitro translation system they developed from embryonic extracts [12]. These extracts passed a useful fidelity test by recapitulating the contribution of individual Sxl-binding sites in vitro. Two significant mechanistic findings emerged from this work. First, although regulation of many other mRNAs appears to be mediated through the poly(A) tail, repression of msl-2 by Sxl is poly(A) independent. The strongest evidence in support of this conclusion was equivalent repression of 3′ adenylated msl-2 mRNA and unadenylated mRNA terminated with cordycepin (to block adenylation during incubation in the extract). Second, the authors showed that a minimal RNA-binding fragment of Sxl (containing little more than the two RRMs) is sufficient to confer nearly normal translational control in vitro. This finding suggests that translational effector function is embedded within the conserved RNA-binding motif, a theme we will return to in the case of Pumilio (below). Bruno: a repressor of oskar mRNA
Specification of both the germline and the posterior body plan relies on a division of the cytoplasm of oocyte and early embryo into two compartments: the ‘privileged’ pole plasm [13], a thin crescent at the posterior extremity, and the ‘bulk’ cytoplasm that comprises the remainder. For the purposes of this review, the ‘privilege’ enjoyed by mRNA occupants of the pole plasm is the right to translation; ‘unlocalized’ mRNA in the bulk cytoplasm is repressed. Progress in understanding the basis of this translational privilege has been hampered by the failure thus far to identify a single pole plasm component that acts directly to localize or translationally activate targets such as oskar or nanos (nos) mRNA. By contrast, a key repressor of the unlocalized mRNA has been identified for each (Fig. 2). Oskar protein accumulates exclusively at the posterior of the oocyte (in the ovary) and the embryo (following fertilization) even though appreciable levels of mRNA are distributed throughout both. This tight spatial restriction to a sub-cellular compartment (i.e. the pole plasm) is essential for http://tig.trends.com
hb
HB TRENDS in Genetics
Fig. 2. The cascade of translational regulation that governs abdominal segmentation, with proteins in upper case, and mRNAs in lower case italic. Repression is mediated by direct action of the RNA-binding proteins Bruno, Smaug (Smg) and Pumilio (Pum). Nanos (NOS) and Brain Tumor (BRAT) are recruited to hunchback (hb) mRNA by PUM. The mechanism of Oskar (Osk)-dependent translational activation is not yet clear. Red T-bar, repression; green arrow, activation; black arrow, translation.
normal development. Overexpression of wild-type osk mRNA in transgenic flies leads to the ectopic accumulation of Osk in the bulk cytoplasm of the embryo, presumably due to titration of a limiting repressor [14,15]. Further evidence pointing to the existence of repression signals in the osk 3′ UTR came from experiments of Ephrussi and Lehmann, who expressed a chimeric mRNA in vivo. One trans-acting component of the osk repression system was identified in a particularly elegant and definitive series of experiments by Macdonald and colleagues [16]. These began with the identification of an 89-kDa ovarian protein, named Bruno, that binds specifically to portions of the osk 3′ UTR in UV cross-linking experiments. Expression of an altered osk mRNA in which all six Bruno-binding sites are mutated causes derepression in the oocyte, proving that the sites collectively mediate regulation. Evidence that Bruno is the factor that acts through these sites in vivo (and not some other protein with similar binding specificity, for example) followed isolation of the gene encoding Bruno. This proved to be arrest (aret), previously identified on the basis of adult viable mutations that interfere with both spermatogenesis and oogenesis [17]. Because oogenesis is blocked relatively early in homozygous mutant animals, it proved impossible to eliminate Bruno function and thereby assess its role in osk mRNA repression, which takes place later in development. But in a sensitized background, Webster et al. [17] showed that the reduced level of Bruno in heterozygous animals is insufficient to repress fully an mRNA bearing wild-type osk 3′ UTR sequences and that a similar mRNA with mutant Bruno-binding sites is insensitive
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to changes in Bruno concentration. Thus, Bruno represses unlocalized osk mRNA in the oocyte. Although little is yet understood of how Bruno acts, it appears to have partners, much as is the case for Smaug and Pumilio (as described below). Bruno contains three RRMs, of which the most C-terminal is sufficient for specific binding. The protein is abundant in ovaries, but absent from early embryos, providing the first hint that another repressor probably acts to block translation of unlocalized osk mRNA in the embryo. Two groups took advantage of the absence of Bruno from embryonic extracts to show that the addition of recombinant protein to such extracts could impose repression on otherwise unregulated mRNA [18,19]. The extent of repression in supplemented embryonic extracts was less than that in ovarian extracts, again suggesting the existence of additional repressors. As a test of this idea, Lie and Macdonald [18] asked whether an mRNA with a synthetic 3′ UTR containing multiple Bruno-binding sites but no other osk-derived sequences can be repressed in vitro. Their results clearly indicate that, in addition to Bruno-binding sites, other signals in the osk 3′ UTR are required for regulation. The mechanism of Bruno-mediated repression remains to be determined. Early experiments suggested that, like msl-2 mRNA (and unlike other maternal mRNAs [20]), the poly(A) tail plays little role in osk regulation. The poly(A) tail of the endogenous mRNA is short and not elongated in vivo upon translational activation. Consistent with the idea that Bruno does not act via the poly(A) tail, Lie and Macdonald found that unadenylated mRNA is repressible in vivo [18]. However, in these experiments the substrate was not terminated with cordycepin to prevent adenylation during incubation in the extract (as described above for Sxl-dependent repression in vitro); the method used to detect adenylation during incubation was relatively insensitive and probably would have missed the addition of short poly(A) tails to a minor subset of substrate molecules; and the instability of uncapped mRNA in the extracts thwarted attempts to determine whether Bruno requires a 5′ cap. These issues have yet to be addressed, and so the mechanism of Bruno action remains unknown. One further line of enquiry suggests that Bruno, similar to Pumilio, acts by nucleating the assembly of a heteromultimeric repression complex. To identify potential Bruno co-factors, Lie and Macdonald performed a two-hybrid screen that led to the isolation of a bZIP protein named Apontic (Apt) [21]. A direct genetic test of the role of Apt in osk regulation was not possible, because oogenesis arrests early in germline clones that lack apt function. But doubly heterozygous apt−/+ aret−/+ females yield embryos with developmental defects diagnostic of ectopic Osk activity, supporting the idea that Bruno and Apt do cooperate in vivo. The argument for cooperation would be significantly stronger if ectopic Osk activity had http://tig.trends.com
been detected directly. In addition, it would be desirable to test the Bruno–Apt interaction genetically, with one or more substitutions in Bruno that specifically disrupt Apt interaction without affecting RNA-binding, for example. Nevertheless, it seems likely that Bruno collaborates with Apt (and perhaps other factors) on osk mRNA to regulate its translation. Smaug: a repressor of nanos mRNA
Much as is the case for oskar, Nos protein accumulates only in the posterior of the early embryo even though nos mRNA is distributed throughout the early embryo. This distribution is not uniform: the mRNA is localized or concentrated in the pole plasm at a higher level than is found in the bulk cytoplasm. Genetic and biochemical experiments show that only the minor fraction of nos mRNA resident in the pole plasm, estimated to be ~4% of total, gives rise to detectable protein [22,23]. Thus, the observed asymmetric distribution of Nos protein must depend either on degradation of Nos protein or on repression of unlocalized nos mRNA in the bulk cytoplasm. Evidence in favor of the latter mechanism was reported by three laboratories. Smibert et al. identified a 120-kDa factor in embryonic extracts that binds to two short hairpins in the 3′ UTR of the nos mRNA. Mutations in the mRNA abolished binding of the factor, dubbed Smaug, resulting in derepression of the unlocalized mRNA [24]. Dahanukar and Wharton came to the same conclusion from a deletion analysis of regulatory signals in the 3′ UTR. They also showed that, in the context of an otherwise unaltered mRNA, either hairpin is sufficient for near normal repression [25]. Gavis et al. also identified one of the hairpins, but missed the other [26], probably because of their use of a nanos–tubulin (nos–tub) chimeric mRNA that is translated with excessive enthusiasm as a result of signals in the tubulin moiety. Smaug was identified independently by purification of the hairpin-binding activity, to allow amino acid sequencing of peptides [27], and by use of the three-hybrid RNA-binding assay in yeast [28]. Smaug recognizes its hairpins using a novel RNA-binding domain [28] that includes a sterile α motif, thought to be involved primarily in mediating protein–protein contacts in other cases [29]. Two lines of evidence show that Smaug-binding is indeed essential for repression. First, isolation of a mutation in the smg gene led to the demonstration that nos mRNA is derepressed in the absence of functional Smaug in vivo [28]. Second, immunodepletion of Smaug from early embryonic abstracts abolished translational repression of appropriate exogenous substrates in embryonic extracts [27]. (These results were qualified by the authors because of their inability to restore repression to the depleted extracts with recombinant protein.) Together, these results show that Smaug is required for repression of the unlocalized nos mRNA. The mechanism of this repression has been investigated in a recent report. Although 96% of the
Review
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nos mRNA in the embryo is repressed in vivo, 30–60% of the total nos mRNA extracted from early embryos was found to be associated with fast-sedimenting material in sucrose gradients [30]. The association is sensitive to addition of EDTA and puromycin, suggesting that the mRNA is in fact incorporated into polysomes. Therefore, at least one aspect of the translational block appears to occur post-initiation. The authors follow up this observation by comparing the translation of otherwise identical mRNAs with nos and tub 3′ UTRs. However, in the absence of controls to demonstrate specific (i.e. Smg-dependent) regulation, these shed no additional light on the mechanism. Nevertheless, it does appear that most of the nos mRNA in the embryo is repressed post-initiation. The question of whether Smaug requires other RNA-bound factors to repress translation has not been fully addressed. Smibert has found that addition of wild-type but not mutant Smaug-binding sites is sufficient to confer regulation on two different reporter mRNAs, one in transgenic embryos and the other in translation extracts [24,27]. However, it is possible that both reporters fortuitously bear binding sites for other essential factors, so it would be inappropriate to conclude that binding of Smaug is sufficient for repression. Indeed, one hint at the existence of another factor comes from the work of Crucs et al. [31], who found that a second stem-loop adjacent to the 5′ Smaug-binding site is required for repression of a chimeric nos–tub mRNA in transgenic embryos. Unfortunately, these experiments are subject to the same caveat regarding the nos–tub chimera outlined above. The role of this second stem-loop in native nos mRNA is untested and therefore unclear. A question that remains outstanding is how Smaug-dependent repression is overcome in the pole plasm. Unfortunately, no trans-acting factor that binds directly to nos mRNA to activate its translation has yet been identified. Smaug protein is uniformly distributed throughout the early embryo and thus present in the pole plasm where nos mRNA is translated [28]. (Note that Smibert et al. [27] claim to see Smaug accumulation preferentially at the posterior pole in later developmental stages, after Smaug has begun to act. This seems unlikely, given the distribution reported by Dahanukar and Wharton [25] with different antibodies and a control for their specificity in situ, smg- embryos.) How then does nos mRNA escape repression by Smaug? One attractive model derived from the observation that the Smaug RNA-binding domain interacts in yeast and in vitro with Oskar [28], which is required (in a genetic sense) for translation of nos mRNA. However, this alluring model did not stand up to a critical test: substitutions in Smaug that substantially reduce its interaction with Oskar without affecting RNA-binding have no effect on Smaug function or embryonic patterning when assayed in transgenic animals (C. Gardner and R. Wharton, unpublished). So vanishes the only extant molecular model for nos mRNA activation. http://tig.trends.com
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Pumilio and its collaborators: repressors of hunchback mRNA
Perhaps the most completely characterized case of translational repression in Drosophila is that of maternal hunchback (hb) mRNA. For some time, it has been clear that differential accumulation of Hb protein along the anterior–posterior axis of the early embryo is driven by events at two similar elements in the 3′ UTR of the mRNA, named Nanos response elements (NREs) [32]. These are necessary and sufficient to confer Pumilio-dependent regulation when tested in three different maternal mRNAs and a chimeric mRNA expressed in the eye imaginal disc [33]. Here we focus on recent developments – the discovery that a large heteromeric repressor complex assembles on the NREs and insights into the mechanisms of repression. Despite their name, the NREs are primarily binding sites for Pumilio [32], a factor known from genetic studies to have an essential role in hb regulation [34]. Pum and fem-3-binding factor [35] are the founding members of the PUF family of RNA-binding proteins, defined by up to eight 36 amino acid tandem repeats. In the X-ray crystal structures of human and fly Pum, these repeats are elaborated into a rainbow arc that constitutes a novel RNA-binding surface [36,37]. A high-resolution co-crystal structure shows that it is the ‘inner’ surface of the Pum arc that interacts with the NRE [38]. As appears to be the case for Sxl, essential translational effector function is embedded within the RNA-binding domain, since expression of this protein fragment is largely sufficient to rescue abdominal segmentation in otherwise pum− embryos [33]. At least part of this effector function involves recruitment of two essential co-factors by residues on the outer surface of the Pum rainbow arc. Gratifyingly, one of these proved to be Nos. Despite clear genetic evidence that Nos is the spatially limiting factor, the mechanism by which it specifically targets hb mRNA for repression escaped definition for an embarrassingly long interval. The mechanism was finally defined by Sonoda, who showed that Nos is recruited into a ternary complex with NRE-bound Pum [39]. Mutational analysis of all three components supports a model in which a small number of low-specificity protein–protein and protein–RNA interactions sum to stabilize the Pum–Nos–NRE ternary complex. Unlike Nos, the existence of the second co-factor recruited by Pum was unanticipated until recently. The studies that led to its isolation were sparked by the genetic and biochemical properties of an unusual mutant of Pum that binds the NRE and recruits Nos normally, yet is ineffective at regulating translation in vivo [33]. Armed with this singly substituted Pum RNA-binding domain, Sonoda isolated Brain Tumor (Brat) in a yeast four-hybrid scheme, looking for factors that bind specifically to the wild-type NRE–Pum–Nos complex, but not to the corresponding complex assembled on the unusual Pum mutant and not to individual components [40]. Brat passed key genetic tests: substitutions of Pum, Nos and Brat that
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block formation of a quaternary complex abrogate regulation of hb mRNA in the early embryo. Brat is a member of a protein family described by one of the least euphonius acronyms to grace molecular biology: RBCC-NHL [41]. Interaction of Brat with the ternary complex is mediated by its NHL domain, a β-propeller motif that provides a large interaction surface. Intriguingly, expression of the Brat NHL domain alone is sufficient to rescue hb regulation in brat− embryos, suggesting that, as is the case for Pum and Sxl, translational effector function is embedded in the same domain that recruits Brat to the RNA. The picture that emerges from the work described above is rather rococo. Apparently, a total of at least 0.62 MDa of protein (assuming binding of one protomer each of Pum, Nos and Brat to each NRE) is loaded on the 3′ UTR of hb to repress its translation in the wild-type embryo. A view of the core complex at atomic resolution is undoubtedly not far off, with the X-ray crystal structures of a Pum-RNA complex [38] and the Brat NHL domain (T. Edwards and A. Aggarwal, unpublished) in hand. How then does this corpulent assembly repress translation? Several lines of evidence suggest that the Pum–Nos–Brat complex can regulate translation by two different mechanisms. One appears to involve NRE-dependent shortening of the poly(A) tail, first observed in endogenous embryonic mRNAs over a decade ago [42]. This has been explored more recently using modified mRNA substrates microinjected into early embryos, which then serve as in vivo test tubes [43]. Based on these experiments, NRE-dependent repression appears to consist of two nearly equal components: a 3–3.5-fold effect that is poly(A)-independent, and a residual 2–2.5-fold effect mediated by the poly(A) tail. The experiments to demonstrate poly(A)-independence were particularly thorough, using both mRNAs incapable of adenylation (because of the presence of a terminal cordycepin References 1 Gray, N.K. and Wickens, M. (1998) Control of translation initiation in animals. Annu. Rev. Cell Dev. Biol. 14, 399–458 2 Johnstone, O. and Lasko, P. (2001) Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu. Rev. Genet. 35, 365–406 3 Niessing, D. et al. (2000) Homeodomain position 54 specifies transcriptional versus translational control by Bicoid. Mol. Cell 5, 395–401 4 Okabe, M. et al. (2001) Translational repression determines a neuronal potential in Drosophila asymmetric cell division. Nature 411, 94–98 5 Sosnowski, B.A. et al. (1989) Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage. Cell 58, 449–459 6 Bell, L.R. et al. (1991) Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65, 229–239 7 Kelley, R.L. et al. (1995) Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell 81, 867–877 http://tig.trends.com
residue or the mutation of the AAUAAA poly(A) addition signal) as well as an mRNA terminated with the stem-loop signal that substitutes for a poly(A) tail on cell-cycle-regulated histone mRNAs. The identity of the second, poly(A)-independent mechanism is unclear, but it probably does not involve interference with the initial step of translation – recognition of the 5′ cap – because the NRE is capable of mediating inhibition of IRES-driven translation in eye imaginal disc cells [33]. In summary, the repression complex seems to act by exerting two relatively small effects that therefore are likely to be difficult to study in vitro – one downstream of cap recognition and one mediated by the poly(A) tail. Summary
From the work described above, two common themes of translational control emerge. First, translational repressors function by engaging multiple binding sites of the mRNA. And second, translational repressors rarely work alone; assembly of large heteromeric repression complexes is usually required. With the possible exception of hb regulation, the complete set of cis-acting factors has probably not yet been identified for any of the examples discussed above. Molecular complexity on the 3′ UTRs of regulated mRNAs is considerable, especially if various combinations of repressors are assembled on different target mRNAs, as has been suggested. The study of translational repressors in Drosophila development has been particularly productive in identifying novel RNA-binding motifs (Pum and Smg). A significant weakness in this (narrowly defined) field has been the elucidation of mechanism, which has been led by studies of mammalian mRNAs such as ferritin and lox-15 [44,45]. Research in the next few years is likely to address this shortcoming, and to focus on the modification of repressor activity by co-factors and other regulators.
8 Bashaw, G.J. and Baker, B.S. (1995) The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal. Development 121, 3245–3258 9 Zhou, S. et al. (1995) Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothioneinlike cysteine cluster. EMBO J. 14, 2884–2895 10 Bashaw, G.J. and Baker, B.S. (1997) The regulation of the Drosophila msl-2 gene reveals a function for Sex-lethal in translational control. Cell 89, 789–798 11 Kelley, R.L. et al. (1997) Sex lethal controls dosage compensation in Drosophila by a non-splicing mechanism. Nature 387, 195–199 12 Gebauer, F. et al. (1999) Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5′ and 3′ UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. 18, 6146–6154 13 Rongo, C. et al. (1997) Germ plasm assembly and germ cell migration in Drosophila. Cold Spring Harb. Symp. Quant. Biol. 62, 1–11
14 Smith, J.L. et al. (1992) Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos. Cell 70, 849–859 15 Ephrussi, A. and Lehmann, R. (1992) Induction of germ cell formation by oskar. Nature 358, 387–392 16 Kim-Ha, J. et al. (1995) Translational regulation of oskar mRNA by Bruno, an ovarian RNAbinding protein, is essential. Cell 81, 403–412 17 Webster, P.J. et al. (1997) Translational repressor bruno plays multiple roles in development and is widely conserved. Genes Dev. 11, 2510–2521 18 Lie, Y.S. and Macdonald, P.M. (1999) Translational regulation of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts. Development 126, 4989–4996 19 Castagnetti, S. et al. (2000) Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development 127, 1063–1068 20 Salles, F.J. et al. (1994) Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266, 1996–1999
Review
TRENDS in Genetics Vol.18 No.11 November 2002
Translational repressors in Drosophila Kellie A. Dean, Aneel K. Aggarwal and Robin P. Wharton Translational regulation is an important aspect of gene regulation, particularly during early development of the fruit fly embryo when transcriptional mechanisms are untenable. Study of pattern formation and dosage compensation has identified several repressors that bind discrete sites in the untranslated portions of target mRNAs. These repressors do not work in isolation – each binds multiple sites in the appropriate mRNA, and the resulting RNA–protein complexes appear to recruit co-repressors by a variety of mechanisms.
which regulate translation of caudal and tramtrack69 mRNAs, respectively [3,4]). Most of the translation factors that pass muster are involved in early development, although the first to be discussed below, Sex-lethal (Sxl), is required for dosage compensation and sex determination throughout the life of the fly. Sxl: a regulator of both splicing and translation
Published online: 24 September 2002
Kellie A. Dean Robin P. Wharton* Howard Hughes Medical Institute, Dept of Molecular Genetics and Microbiology, Box 3657, Duke University Medical Center, Durham, NC 27710, USA. *e-mail: [email protected] Aneel K. Aggarwal Structural Biology Program, Dept of Physiology and Biophysics, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA.
Translational regulation of mRNA is a widespread strategy that is used to govern fundamental cellular processes (e.g. iron homeostasis in mammals), as well as esoteric events such as the sperm–oocyte switch in the hermaphroditic nematode Caenorhabditis elegans (reviewed in [1,2]). During stages of the life cycle when the genome is transcriptionally inaccessible, translational control (and other post-transcriptional mechanisms) have predominant roles; for example, during the DNA compaction of spermatogenesis or in anucleate erythrocytes. Establishment of the body plan in the Drosophila embryo takes place during one such period of transcriptional reticence. The embryonic pattern derives from localized maternal products that are deposited in the oocyte, which itself is arrested in meiosis and transcriptionally inert. Subsequent patterning is elaborated during the first few hours of embryonic development when the nuclei are in manic, 10-min cleavage cycles that are incompatible with extensive transcription. As a consequence of these restrictions, translational mechanisms have a significant role in early pattern formation. Progress in the field has been led by study of inhibitors; in the past few years, the early embryo has yielded six novel translational repressors and two novel RNA-binding motifs. This review is not intended to be comprehensive, even among Drosophila proteins. Rather, it is limited to a discussion of translational repressors defined by three criteria. First, in addition to discordant accumulation of mRNA and protein in two different cell types (or subcellular compartments), repression is conferred by sequences in the untranslated regions of the mRNA. Second, one or more factors bind to discrete sites in the mRNA. And third, mutations in the gene encoding the binding factor have the same biological and molecular consequences as mutations in its binding site(s) within the mRNA. These criteria avoid the possibility that apparent translational control is in fact the result of differential protein degradation. They also narrowly exclude some interesting factors (such as Bicoid and Musashi, http://tig.trends.com
Sxl is a master regulator, required in females and obligatorily absent in males. It was originally shown to be a splicing factor during sex determination, governing the production of sex-specific mRNA isoforms at the head of a cascade of regulated splicing events. In this guise, Sxl recognizes uridylate-rich sequences near splicing sites using a pair of RRMs (RNA-recognition motifs) [5,6]. Sxl also controls dosage compensation, and it was natural to assume it would do so by similar methods. However, two groups have shown independently that Sxl has a more interesting role than initially suspected. One of the critical effectors of dosage compensation is Msl-2 protein, which accumulates in males but not in females although both contain its mRNA [7–9]. However, the structure of the mRNA is different in each sex: the female mRNA isoform bears sequences in the 5′ untranslated region (UTR) that are excised as an intron to generate the male isoform. Within this intron are two Sxl-binding sites, and Sxl activity is required to block excision of the intron in females (Fig. 1). The key experiment indicting Sxl as a translational repressor was mutation of the splice acceptor and donor sites that define the intron. Bashaw and Baker [10] drove expression of a suitably modified msl-2 gene with a strong constitutive promoter in both cultured S2 cells and transgenic flies; Kuroda and colleagues pursued the more prudent course of modifying an otherwise wild type msl-2 transgene (thereby relying on endogenous transcriptional and processing signals to generate normal levels of mRNA precursor) [11]. Happily, both groups reached the same conclusion: even if the intron is forcibly included, binding of Sxl represses translation of the mRNA. Thus, Sxl prevents excision of its binding sites in the 5′ UTR so that it can subsequently repress translation from the selfsame sites (in females). Further work uncovered a complexity shared by all of the repressors described in this review – full repression in vivo requires occupancy of multiple binding sites in the mRNA. In the case of Sxl and msl-2 mRNA, one of the binding sites in the 5′ UTR mediates most of the repression, but the nearby site, as well as a cluster of sites near the 3′ terminus of the mRNA, contribute significantly.
0168-9525/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(02)02792-0
Review
TRENDS in Genetics Vol.18 No.11 November 2002
573
osk
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nos
NOS
Fig. 1. Regulation of msl-2 mRNA by Sxl. In females, Sxl binds to sites in the 5′ UTR of the pre-mRNA, preventing their excision so that cytoplasmic Sxl can subsequently repress translation. In males, the 5′ intron is excised in the absence of Sxl and the mRNA is not repressed.
PUM BRAT SMG
Gebauer and colleagues have begun to explore the mechanism of Sxl-dependent repression using an in vitro translation system they developed from embryonic extracts [12]. These extracts passed a useful fidelity test by recapitulating the contribution of individual Sxl-binding sites in vitro. Two significant mechanistic findings emerged from this work. First, although regulation of many other mRNAs appears to be mediated through the poly(A) tail, repression of msl-2 by Sxl is poly(A) independent. The strongest evidence in support of this conclusion was equivalent repression of 3′ adenylated msl-2 mRNA and unadenylated mRNA terminated with cordycepin (to block adenylation during incubation in the extract). Second, the authors showed that a minimal RNA-binding fragment of Sxl (containing little more than the two RRMs) is sufficient to confer nearly normal translational control in vitro. This finding suggests that translational effector function is embedded within the conserved RNA-binding motif, a theme we will return to in the case of Pumilio (below). Bruno: a repressor of oskar mRNA
Specification of both the germline and the posterior body plan relies on a division of the cytoplasm of oocyte and early embryo into two compartments: the ‘privileged’ pole plasm [13], a thin crescent at the posterior extremity, and the ‘bulk’ cytoplasm that comprises the remainder. For the purposes of this review, the ‘privilege’ enjoyed by mRNA occupants of the pole plasm is the right to translation; ‘unlocalized’ mRNA in the bulk cytoplasm is repressed. Progress in understanding the basis of this translational privilege has been hampered by the failure thus far to identify a single pole plasm component that acts directly to localize or translationally activate targets such as oskar or nanos (nos) mRNA. By contrast, a key repressor of the unlocalized mRNA has been identified for each (Fig. 2). Oskar protein accumulates exclusively at the posterior of the oocyte (in the ovary) and the embryo (following fertilization) even though appreciable levels of mRNA are distributed throughout both. This tight spatial restriction to a sub-cellular compartment (i.e. the pole plasm) is essential for http://tig.trends.com
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Fig. 2. The cascade of translational regulation that governs abdominal segmentation, with proteins in upper case, and mRNAs in lower case italic. Repression is mediated by direct action of the RNA-binding proteins Bruno, Smaug (Smg) and Pumilio (Pum). Nanos (NOS) and Brain Tumor (BRAT) are recruited to hunchback (hb) mRNA by PUM. The mechanism of Oskar (Osk)-dependent translational activation is not yet clear. Red T-bar, repression; green arrow, activation; black arrow, translation.
normal development. Overexpression of wild-type osk mRNA in transgenic flies leads to the ectopic accumulation of Osk in the bulk cytoplasm of the embryo, presumably due to titration of a limiting repressor [14,15]. Further evidence pointing to the existence of repression signals in the osk 3′ UTR came from experiments of Ephrussi and Lehmann, who expressed a chimeric mRNA in vivo. One trans-acting component of the osk repression system was identified in a particularly elegant and definitive series of experiments by Macdonald and colleagues [16]. These began with the identification of an 89-kDa ovarian protein, named Bruno, that binds specifically to portions of the osk 3′ UTR in UV cross-linking experiments. Expression of an altered osk mRNA in which all six Bruno-binding sites are mutated causes derepression in the oocyte, proving that the sites collectively mediate regulation. Evidence that Bruno is the factor that acts through these sites in vivo (and not some other protein with similar binding specificity, for example) followed isolation of the gene encoding Bruno. This proved to be arrest (aret), previously identified on the basis of adult viable mutations that interfere with both spermatogenesis and oogenesis [17]. Because oogenesis is blocked relatively early in homozygous mutant animals, it proved impossible to eliminate Bruno function and thereby assess its role in osk mRNA repression, which takes place later in development. But in a sensitized background, Webster et al. [17] showed that the reduced level of Bruno in heterozygous animals is insufficient to repress fully an mRNA bearing wild-type osk 3′ UTR sequences and that a similar mRNA with mutant Bruno-binding sites is insensitive
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to changes in Bruno concentration. Thus, Bruno represses unlocalized osk mRNA in the oocyte. Although little is yet understood of how Bruno acts, it appears to have partners, much as is the case for Smaug and Pumilio (as described below). Bruno contains three RRMs, of which the most C-terminal is sufficient for specific binding. The protein is abundant in ovaries, but absent from early embryos, providing the first hint that another repressor probably acts to block translation of unlocalized osk mRNA in the embryo. Two groups took advantage of the absence of Bruno from embryonic extracts to show that the addition of recombinant protein to such extracts could impose repression on otherwise unregulated mRNA [18,19]. The extent of repression in supplemented embryonic extracts was less than that in ovarian extracts, again suggesting the existence of additional repressors. As a test of this idea, Lie and Macdonald [18] asked whether an mRNA with a synthetic 3′ UTR containing multiple Bruno-binding sites but no other osk-derived sequences can be repressed in vitro. Their results clearly indicate that, in addition to Bruno-binding sites, other signals in the osk 3′ UTR are required for regulation. The mechanism of Bruno-mediated repression remains to be determined. Early experiments suggested that, like msl-2 mRNA (and unlike other maternal mRNAs [20]), the poly(A) tail plays little role in osk regulation. The poly(A) tail of the endogenous mRNA is short and not elongated in vivo upon translational activation. Consistent with the idea that Bruno does not act via the poly(A) tail, Lie and Macdonald found that unadenylated mRNA is repressible in vivo [18]. However, in these experiments the substrate was not terminated with cordycepin to prevent adenylation during incubation in the extract (as described above for Sxl-dependent repression in vitro); the method used to detect adenylation during incubation was relatively insensitive and probably would have missed the addition of short poly(A) tails to a minor subset of substrate molecules; and the instability of uncapped mRNA in the extracts thwarted attempts to determine whether Bruno requires a 5′ cap. These issues have yet to be addressed, and so the mechanism of Bruno action remains unknown. One further line of enquiry suggests that Bruno, similar to Pumilio, acts by nucleating the assembly of a heteromultimeric repression complex. To identify potential Bruno co-factors, Lie and Macdonald performed a two-hybrid screen that led to the isolation of a bZIP protein named Apontic (Apt) [21]. A direct genetic test of the role of Apt in osk regulation was not possible, because oogenesis arrests early in germline clones that lack apt function. But doubly heterozygous apt−/+ aret−/+ females yield embryos with developmental defects diagnostic of ectopic Osk activity, supporting the idea that Bruno and Apt do cooperate in vivo. The argument for cooperation would be significantly stronger if ectopic Osk activity had http://tig.trends.com
been detected directly. In addition, it would be desirable to test the Bruno–Apt interaction genetically, with one or more substitutions in Bruno that specifically disrupt Apt interaction without affecting RNA-binding, for example. Nevertheless, it seems likely that Bruno collaborates with Apt (and perhaps other factors) on osk mRNA to regulate its translation. Smaug: a repressor of nanos mRNA
Much as is the case for oskar, Nos protein accumulates only in the posterior of the early embryo even though nos mRNA is distributed throughout the early embryo. This distribution is not uniform: the mRNA is localized or concentrated in the pole plasm at a higher level than is found in the bulk cytoplasm. Genetic and biochemical experiments show that only the minor fraction of nos mRNA resident in the pole plasm, estimated to be ~4% of total, gives rise to detectable protein [22,23]. Thus, the observed asymmetric distribution of Nos protein must depend either on degradation of Nos protein or on repression of unlocalized nos mRNA in the bulk cytoplasm. Evidence in favor of the latter mechanism was reported by three laboratories. Smibert et al. identified a 120-kDa factor in embryonic extracts that binds to two short hairpins in the 3′ UTR of the nos mRNA. Mutations in the mRNA abolished binding of the factor, dubbed Smaug, resulting in derepression of the unlocalized mRNA [24]. Dahanukar and Wharton came to the same conclusion from a deletion analysis of regulatory signals in the 3′ UTR. They also showed that, in the context of an otherwise unaltered mRNA, either hairpin is sufficient for near normal repression [25]. Gavis et al. also identified one of the hairpins, but missed the other [26], probably because of their use of a nanos–tubulin (nos–tub) chimeric mRNA that is translated with excessive enthusiasm as a result of signals in the tubulin moiety. Smaug was identified independently by purification of the hairpin-binding activity, to allow amino acid sequencing of peptides [27], and by use of the three-hybrid RNA-binding assay in yeast [28]. Smaug recognizes its hairpins using a novel RNA-binding domain [28] that includes a sterile α motif, thought to be involved primarily in mediating protein–protein contacts in other cases [29]. Two lines of evidence show that Smaug-binding is indeed essential for repression. First, isolation of a mutation in the smg gene led to the demonstration that nos mRNA is derepressed in the absence of functional Smaug in vivo [28]. Second, immunodepletion of Smaug from early embryonic abstracts abolished translational repression of appropriate exogenous substrates in embryonic extracts [27]. (These results were qualified by the authors because of their inability to restore repression to the depleted extracts with recombinant protein.) Together, these results show that Smaug is required for repression of the unlocalized nos mRNA. The mechanism of this repression has been investigated in a recent report. Although 96% of the
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nos mRNA in the embryo is repressed in vivo, 30–60% of the total nos mRNA extracted from early embryos was found to be associated with fast-sedimenting material in sucrose gradients [30]. The association is sensitive to addition of EDTA and puromycin, suggesting that the mRNA is in fact incorporated into polysomes. Therefore, at least one aspect of the translational block appears to occur post-initiation. The authors follow up this observation by comparing the translation of otherwise identical mRNAs with nos and tub 3′ UTRs. However, in the absence of controls to demonstrate specific (i.e. Smg-dependent) regulation, these shed no additional light on the mechanism. Nevertheless, it does appear that most of the nos mRNA in the embryo is repressed post-initiation. The question of whether Smaug requires other RNA-bound factors to repress translation has not been fully addressed. Smibert has found that addition of wild-type but not mutant Smaug-binding sites is sufficient to confer regulation on two different reporter mRNAs, one in transgenic embryos and the other in translation extracts [24,27]. However, it is possible that both reporters fortuitously bear binding sites for other essential factors, so it would be inappropriate to conclude that binding of Smaug is sufficient for repression. Indeed, one hint at the existence of another factor comes from the work of Crucs et al. [31], who found that a second stem-loop adjacent to the 5′ Smaug-binding site is required for repression of a chimeric nos–tub mRNA in transgenic embryos. Unfortunately, these experiments are subject to the same caveat regarding the nos–tub chimera outlined above. The role of this second stem-loop in native nos mRNA is untested and therefore unclear. A question that remains outstanding is how Smaug-dependent repression is overcome in the pole plasm. Unfortunately, no trans-acting factor that binds directly to nos mRNA to activate its translation has yet been identified. Smaug protein is uniformly distributed throughout the early embryo and thus present in the pole plasm where nos mRNA is translated [28]. (Note that Smibert et al. [27] claim to see Smaug accumulation preferentially at the posterior pole in later developmental stages, after Smaug has begun to act. This seems unlikely, given the distribution reported by Dahanukar and Wharton [25] with different antibodies and a control for their specificity in situ, smg- embryos.) How then does nos mRNA escape repression by Smaug? One attractive model derived from the observation that the Smaug RNA-binding domain interacts in yeast and in vitro with Oskar [28], which is required (in a genetic sense) for translation of nos mRNA. However, this alluring model did not stand up to a critical test: substitutions in Smaug that substantially reduce its interaction with Oskar without affecting RNA-binding have no effect on Smaug function or embryonic patterning when assayed in transgenic animals (C. Gardner and R. Wharton, unpublished). So vanishes the only extant molecular model for nos mRNA activation. http://tig.trends.com
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Pumilio and its collaborators: repressors of hunchback mRNA
Perhaps the most completely characterized case of translational repression in Drosophila is that of maternal hunchback (hb) mRNA. For some time, it has been clear that differential accumulation of Hb protein along the anterior–posterior axis of the early embryo is driven by events at two similar elements in the 3′ UTR of the mRNA, named Nanos response elements (NREs) [32]. These are necessary and sufficient to confer Pumilio-dependent regulation when tested in three different maternal mRNAs and a chimeric mRNA expressed in the eye imaginal disc [33]. Here we focus on recent developments – the discovery that a large heteromeric repressor complex assembles on the NREs and insights into the mechanisms of repression. Despite their name, the NREs are primarily binding sites for Pumilio [32], a factor known from genetic studies to have an essential role in hb regulation [34]. Pum and fem-3-binding factor [35] are the founding members of the PUF family of RNA-binding proteins, defined by up to eight 36 amino acid tandem repeats. In the X-ray crystal structures of human and fly Pum, these repeats are elaborated into a rainbow arc that constitutes a novel RNA-binding surface [36,37]. A high-resolution co-crystal structure shows that it is the ‘inner’ surface of the Pum arc that interacts with the NRE [38]. As appears to be the case for Sxl, essential translational effector function is embedded within the RNA-binding domain, since expression of this protein fragment is largely sufficient to rescue abdominal segmentation in otherwise pum− embryos [33]. At least part of this effector function involves recruitment of two essential co-factors by residues on the outer surface of the Pum rainbow arc. Gratifyingly, one of these proved to be Nos. Despite clear genetic evidence that Nos is the spatially limiting factor, the mechanism by which it specifically targets hb mRNA for repression escaped definition for an embarrassingly long interval. The mechanism was finally defined by Sonoda, who showed that Nos is recruited into a ternary complex with NRE-bound Pum [39]. Mutational analysis of all three components supports a model in which a small number of low-specificity protein–protein and protein–RNA interactions sum to stabilize the Pum–Nos–NRE ternary complex. Unlike Nos, the existence of the second co-factor recruited by Pum was unanticipated until recently. The studies that led to its isolation were sparked by the genetic and biochemical properties of an unusual mutant of Pum that binds the NRE and recruits Nos normally, yet is ineffective at regulating translation in vivo [33]. Armed with this singly substituted Pum RNA-binding domain, Sonoda isolated Brain Tumor (Brat) in a yeast four-hybrid scheme, looking for factors that bind specifically to the wild-type NRE–Pum–Nos complex, but not to the corresponding complex assembled on the unusual Pum mutant and not to individual components [40]. Brat passed key genetic tests: substitutions of Pum, Nos and Brat that
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block formation of a quaternary complex abrogate regulation of hb mRNA in the early embryo. Brat is a member of a protein family described by one of the least euphonius acronyms to grace molecular biology: RBCC-NHL [41]. Interaction of Brat with the ternary complex is mediated by its NHL domain, a β-propeller motif that provides a large interaction surface. Intriguingly, expression of the Brat NHL domain alone is sufficient to rescue hb regulation in brat− embryos, suggesting that, as is the case for Pum and Sxl, translational effector function is embedded in the same domain that recruits Brat to the RNA. The picture that emerges from the work described above is rather rococo. Apparently, a total of at least 0.62 MDa of protein (assuming binding of one protomer each of Pum, Nos and Brat to each NRE) is loaded on the 3′ UTR of hb to repress its translation in the wild-type embryo. A view of the core complex at atomic resolution is undoubtedly not far off, with the X-ray crystal structures of a Pum-RNA complex [38] and the Brat NHL domain (T. Edwards and A. Aggarwal, unpublished) in hand. How then does this corpulent assembly repress translation? Several lines of evidence suggest that the Pum–Nos–Brat complex can regulate translation by two different mechanisms. One appears to involve NRE-dependent shortening of the poly(A) tail, first observed in endogenous embryonic mRNAs over a decade ago [42]. This has been explored more recently using modified mRNA substrates microinjected into early embryos, which then serve as in vivo test tubes [43]. Based on these experiments, NRE-dependent repression appears to consist of two nearly equal components: a 3–3.5-fold effect that is poly(A)-independent, and a residual 2–2.5-fold effect mediated by the poly(A) tail. The experiments to demonstrate poly(A)-independence were particularly thorough, using both mRNAs incapable of adenylation (because of the presence of a terminal cordycepin References 1 Gray, N.K. and Wickens, M. (1998) Control of translation initiation in animals. Annu. Rev. Cell Dev. Biol. 14, 399–458 2 Johnstone, O. and Lasko, P. (2001) Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu. Rev. Genet. 35, 365–406 3 Niessing, D. et al. (2000) Homeodomain position 54 specifies transcriptional versus translational control by Bicoid. Mol. Cell 5, 395–401 4 Okabe, M. et al. (2001) Translational repression determines a neuronal potential in Drosophila asymmetric cell division. Nature 411, 94–98 5 Sosnowski, B.A. et al. (1989) Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage. Cell 58, 449–459 6 Bell, L.R. et al. (1991) Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65, 229–239 7 Kelley, R.L. et al. (1995) Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell 81, 867–877 http://tig.trends.com
residue or the mutation of the AAUAAA poly(A) addition signal) as well as an mRNA terminated with the stem-loop signal that substitutes for a poly(A) tail on cell-cycle-regulated histone mRNAs. The identity of the second, poly(A)-independent mechanism is unclear, but it probably does not involve interference with the initial step of translation – recognition of the 5′ cap – because the NRE is capable of mediating inhibition of IRES-driven translation in eye imaginal disc cells [33]. In summary, the repression complex seems to act by exerting two relatively small effects that therefore are likely to be difficult to study in vitro – one downstream of cap recognition and one mediated by the poly(A) tail. Summary
From the work described above, two common themes of translational control emerge. First, translational repressors function by engaging multiple binding sites of the mRNA. And second, translational repressors rarely work alone; assembly of large heteromeric repression complexes is usually required. With the possible exception of hb regulation, the complete set of cis-acting factors has probably not yet been identified for any of the examples discussed above. Molecular complexity on the 3′ UTRs of regulated mRNAs is considerable, especially if various combinations of repressors are assembled on different target mRNAs, as has been suggested. The study of translational repressors in Drosophila development has been particularly productive in identifying novel RNA-binding motifs (Pum and Smg). A significant weakness in this (narrowly defined) field has been the elucidation of mechanism, which has been led by studies of mammalian mRNAs such as ferritin and lox-15 [44,45]. Research in the next few years is likely to address this shortcoming, and to focus on the modification of repressor activity by co-factors and other regulators.
8 Bashaw, G.J. and Baker, B.S. (1995) The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal. Development 121, 3245–3258 9 Zhou, S. et al. (1995) Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothioneinlike cysteine cluster. EMBO J. 14, 2884–2895 10 Bashaw, G.J. and Baker, B.S. (1997) The regulation of the Drosophila msl-2 gene reveals a function for Sex-lethal in translational control. Cell 89, 789–798 11 Kelley, R.L. et al. (1997) Sex lethal controls dosage compensation in Drosophila by a non-splicing mechanism. Nature 387, 195–199 12 Gebauer, F. et al. (1999) Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5′ and 3′ UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. 18, 6146–6154 13 Rongo, C. et al. (1997) Germ plasm assembly and germ cell migration in Drosophila. Cold Spring Harb. Symp. Quant. Biol. 62, 1–11
14 Smith, J.L. et al. (1992) Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos. Cell 70, 849–859 15 Ephrussi, A. and Lehmann, R. (1992) Induction of germ cell formation by oskar. Nature 358, 387–392 16 Kim-Ha, J. et al. (1995) Translational regulation of oskar mRNA by Bruno, an ovarian RNAbinding protein, is essential. Cell 81, 403–412 17 Webster, P.J. et al. (1997) Translational repressor bruno plays multiple roles in development and is widely conserved. Genes Dev. 11, 2510–2521 18 Lie, Y.S. and Macdonald, P.M. (1999) Translational regulation of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts. Development 126, 4989–4996 19 Castagnetti, S. et al. (2000) Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development 127, 1063–1068 20 Salles, F.J. et al. (1994) Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266, 1996–1999