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Diversity in translational regulation
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Diversity in translational regulation Paul Macdonald Translational control of individual mRNAs relies on cis-regulatory elements, which are often found in the 3′ untranslated region. The best characterized of these regulate cytoplasmic polyadenylation, and much of this process can now be defined in terms of molecular interactions, protein modifications and their consequences. Biochemical and genetic approaches have advanced the understanding of the many instances of translational regulation that are crucial for body patterning in Drosophila. For example, in vitro translation systems have been used to study the regulatory mechanisms, and genetic interactions have been instrumental in establishing a link between a regulatory factor and a component of the translational apparatus. Although most examples of control are thought to affect the initiation of translation, two classes of regulatory factors, one a protein and one a short non-coding RNA now appear to inhibit protein synthesis during elongation. Diversity seems to be a central feature of translational control, both in the mechanisms themselves and in the situations where this form of regulation is used. Addresses Institute for Cellular and Molecular Biology, Section of Molecular Cell and Developmental Biology, The University of Texas at Austin, 2500 Speedway, Austin, Texas 78712-1095, USA; e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:326–331 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations IRESes internal ribosome entry sites PABP poly(A)-binding protein UTR untranslated region
Introduction Control of gene expression persists after transcription, when a diverse spectrum of processes that impact upon the distribution or activity of mRNAs comes into play. The most fundamental of these, given the existence of mRNAs as an intermediate in making proteins from genes, is translation. A decision of whether to translate an mRNA may be applied non-specifically, through global effects on the translation machinery. One of the most important and extensively characterized examples centers on the limiting initiation factor eIF4E, a focal point for regulation in a wide range of cell types [1]. Alternatively, features of individual mRNAs can impose mRNA-specific forms of control. In this review I describe recent progress in understanding translational regulation, focusing on the control mechanisms which are tailored to act on specific mRNAs. In most of the examples described here, the relevant elements of translational regulation are contained within the 3′ untranslated region (UTR), but despite this similarity, the ways in which these elements influence translation can differ substantially (Figure 1).
Translational control and cytoplasmic polyadenylation Many maternal mRNAs are transcribed long before they are translated. For at least some of these mRNAs, such as the c-mos mRNA of Xenopus, the onset of translation plays a crucial role in development of the oocyte [2,3]. For c-mos, and some other mRNAs, translational activation is accompanied by cytoplasmic polyadenylation [4]. This process involves the interplay between a regulatory element (the CPE) in the 3′ UTR of affected mRNAs [4], CPEB, a protein that binds to CPEs [5], and other factors. Oddly enough, CPEB mediates both repression and activation of translation [6••], and recent progress in defining the changing interactions of CPEB has provided an explanation for its opposing behaviors [7•]. During repression CPEB binds to Maskin, a protein isolated as a binding partner for CPEB. Maskin, in turn binds to the translation initiation factor eIF4E (the factor that binds specifically to the 5′ cap). Inclusion of eIF4E in this complex prevents its interaction with eIF4G, another component of the cap-binding complex. This interaction is required to bring the 40S ribosomal subunit to the mRNA. A lingering question concerns the inhibition of eIF4E: what prevents repression from acting in trans, with Maskin at the 3′ UTR of one mRNA molecule inactivating eIF4E bound to the cap of another mRNA? This issue is common to many examples of 3′ UTR-mediated translational regulation, and the answer probably lies in the nature of mRNA packaging in the cytoplasm, which may ensure that the two ends of a single mRNA are in close proximity to each other (circularization is possible under some circumstances [8]) or possibly packaged with coregulated mRNAs in particles. Reversal of CPEB-mediated repression is triggered by phosphorylation of CPEB at Ser174 by the Eg2 kinase [9••]. This change in charge enhances binding of CPEB to the component of the protein complex that adds poly(A) tails to mRNAs [10••], strengthening earlier evidence implicating this complex in cytoplasmic polyadenylation events [11]. Thus, the modification of CPEB recruits the polyadenylation machinery and elongates the poly(A) tail. This leads to the question: how does polyadenylation activate translation? Disruption of the Maskin–eIF4E complex takes place at the same time as elongation of the poly(A) tail, but how this happens is not known. Two answers have been proposed for further stimulation of translation. One invokes a 5′ cap modification that occurs in response to elongation of the poly(A) tail and enhances translation efficiency during oocyte maturation [12]. The second relies on an additional set of molecular interactions defined using in vitro experimental systems. Long poly(A) tails are bound efficiently by the poly(A)-binding protein (PABP). Because PABP binds to eIF4G, the association of PABP with mRNAs bearing long poly(A) tails can help to recruit eIF4G for binding with eIF4E at the 5′ cap and thus
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Figure 1
(a) Repression c-mos mRNA
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Current Opinion in Cell Biology
Variations in mechanisms of translational repression by factors that bind to specific control elements in 3′ UTRs of regulated mRNAs. (a) Control of c-mos translation, both repression and activation, requires CPEB. Different interactions of CPEB and Maskin determine whether translation is allowed. In this example, activation involves extension of the poly(A) tail. See text for details. (b) Repression of oskar mRNA by Bruno does not involve either the cap or the poly(A) tail, and so Bruno (Bru) must inhibit some other step, such as assembly of the complete ribosome on the mRNA [19•]. Additional 3′ UTR-binding proteins, including Apontic (Apt) [50] and possibly p50 [51], contribute to repression. (c) Repression by proteins of the Puf and Nanos families. For the Drosophila hunchback mRNA, the Puf family member Pumilio and Nanos bind to the regulatory sequences (NREs) in a ternary complex, with the Pumilio–Nanos interaction only detected in the presence of the NREs [29•]. In contrast, the C. elegans FBF (fem-3-binding factor) and Nanos-3 proteins, which repress translation of the fem-3 mRNA, can bind to one another independently of the RNA [30]. Other Nanos family members in C. elegans do not detectably interact with FBF in the absence of the
RNA but are required for the same developmental process, a switch from spermatogenesis to oogenesis in hermaphrodites. Thus, it is possible that Puf–Nanos interactions other than Pumilio–Nanos can occur only in the presence of the RNA control elements. Repression of hunchback mRNA by Pumilio and Nanos is correlated with deadenylation [52], although the details of how this effect leads to inhibition of translation for this mRNA are not known. (d) Control at the level of elongation. The Drosophila nanos mRNA is repressed by the binding of Smaug (Smg), and possibly another protein, to the 3′ UTR [32•,33,53], whereas C. elegans lin-14 mRNA is repressed through a probable interaction between its 3′ UTR and the lin-4 regulatory RNA [38–40]. In both cases repression does not substantially alter the fraction of the mRNA associated with polyribosomes [35•,40•]. In Drosophila, the Bicaudal (Bic) protein, which appears to be required for repression of nanos mRNA translation, is the homolog of a component of the nascentpolypeptide-associated complex [37•], and thus may contribute to repression through binding to nascent Nanos protein, again suggesting that repression occurs during elongation.
promote translation [13,14]. However, the low level of PABP in Xenopus oocytes [15] casts doubt on the relevance of the eIF4G–PABP interaction in this setting. Two recent reports reopen this issue. In one set of experiments PABP was targeted to specific mRNAs in Xenopus oocytes using a foreign RNA-binding domain and cognate binding site [16••]. The interaction stimulated translation, apparently through binding
to eIF4G, demonstrating the potential for a stimulatory role of PABP on endogenous mRNAs. In the second set of experiments, expression in Xenopus oocytes of a mutant form of eIF4G unable to interact with PABP had a modest dominant inhibitory effect on translation of polyadenylated mRNAs, including c-mos, and a more substantial effect on oocyte maturation [17••]. Thus, there is now evidence supporting a role
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for the poly(A) tail in recruiting PABP for stimulation of c-mos translation, and it may be that polyadenylation contributes to translational activation through multiple pathways.
Translational control as a means to restrict spatial patterns of protein expression Translational control events limit not only the timing of maternal mRNA expression but also where certain mRNAs are allowed to be translated. This phenomenon is most striking in oogenesis and early embryogenesis of Drosophila. Multiple translational regulatory events help to restrict the spatial expression of proteins that act as patterning determinants and to organize the body pattern of the embryo. The number of mRNAs and proteins known to be involved is now quite large, and a complete description of these events is beyond the scope of this review (see [18] for a recent and comprehensive account). Instead, I will highlight a few recent advances that are likely to have a significant impact on future work or resolve open issues. One recent advance is the development of Drosophilabased in vitro translation systems that reproduce translational regulation using exogenous mRNAs [19•–21•]. These systems contain all the required regulatory factors and offer the promise of dissecting translational control events at the biochemical level. Two of these reports [19•,20•] focus on the repression of oskar mRNA by Bruno, an essential regulatory event that helps ensure that the Oskar protein will be present at high levels only at the posterior pole of the oocyte [22], and the in vitro system has been used to show that repression involves neither the 5′ cap nor the poly(A) tail [19•]. A second advance is the demonstration of a functional interaction between Vasa and the translation factor IF2 [23••]. Vasa is an RNA helicase [24] required for the efficient translation of multiple proteins during oogenesis, including Oskar and Gurken [18]. IF2, also known as eIF5B, is required for joining of the ribosomal subunits [25] and may have additional functions during translation [26]. The Vasa–IF2(eIF5B) interaction provides, at last, a compelling link in flies between a genetically defined regulatory molecule and a general translation factor. Exploring and extending the Vasa–IF2(eIF5B) interaction should provide significant new insights into how regulatory molecules affect the functioning of the translation apparatus. A third notable advance concerns the translational repression of hunchback mRNA, an essential step in posterior body patterning. Nanos, the genetically defined posterior patterning determinant, has long been known to act in this example of repression [27], but its role has been obscure, with no evidence of a direct interaction with either the NREs (the sequences in hunchback mRNA that mediate repression) or Pumilio, the protein that binds specifically to the NREs [28]. Now all three components — Nanos, Pumilio and the NREs — have been shown to form a ternary complex, with the RNA component being required
to bring the two proteins together [29•]. This result contrasts with a similar situation in Caenorhabditis elegans, where two proteins closely related to Nanos and Pumilio interact with each other independently of RNA [30•]. Proteins related to Pumilio, the Puf family [31], are present in organisms ranging from yeast to humans, and it remains to be seen which, if either, of the fly and worm models will serve as the prototype for Puf interactions elsewhere. A final example from Drosophila embryogenesis involves the nanos mRNA, whose translation is repressed throughout much of the embryo by binding of Smaug protein to SREs (or Smaug recognition elements) in the nanos 3′ UTR [32•,33]. A distinct element is also involved and presumably binds to an as yet unidentified factor [34]. Translation only occurs at the posterior pole in a process dependent on a number of genes, including oskar and vasa [35]. Surprisingly, the presence of nanos mRNA in polysomal fractions persists even in oskar and vasa mutants [36•]. Thus, repression appears to be exerted after initiation of translation. Notably, a similar model had already been proposed on the basis of the characterization of the bicaudal gene [37•]. bicaudal mutants allow ectopic translation of nanos mRNA, presumably because of defects in repression of nanos translation. The bicaudal gene encodes a subunit of the nascent-polypeptide-associated complex, which binds to polypeptides as they emerge from the ribosome. Thus, if failure of the complex to bind to the nascent Nanos protein is responsible for the defect in repression, then repression must occur during elongation.
Heterochrony in C. elegans Intervention during the elongation phase of translation has also been implicated in a peculiar form of regulation highlighted by certain heterochronic mutants of C. elegans. Heterochronic mutants affect the timing of developmental events during larval stages of growth. Two of the heterchronic genes, lin-4 and the recently identified let-7, do not themselves encode proteins [38,39]; instead, their products are short cytoplasmic RNAs, each of which is largely complementary to sequences in the 3′ UTRs of overlapping sets of other heterochronic genes (such as lin-14). Where tested, loss of the complementary sequences mimics the effects of mutating the short RNAs, leading to reduced levels of the proteins encoded by the target mRNAs [38–40]. The effect on protein levels cannot be explained by altered mRNA stability, and so this appears to be a novel form of translational regulation. Recent studies have addressed the question of which translational step is the target for lin-4-dependent repression of lin-14 translation [42•]. Results very similar to those obtained with nanos mRNA, showed that repression is achieved with no obvious loss of polysomal association. Thus, the short RNAs appear to affect translation after initiation or to affect the stability of the nascent protein. Remarkably, one of the short RNAs, let-7, is present in a wide range of animal species [41••]. The timing of expression of the let-7 RNA in some of these species suggests a conserved role in temporal control of developmental events,
Diversity in translational regulation Macdonald
and it seems likely that a molecular function in translational control should also be conserved.
How widespread is translational regulation? Discussions of translational regulation often turn to the question of whether its use is limited to certain cell types or whether it has a broader role. At one level the answer is already clear, as control mechanisms that govern the translational machinery have been shown to act in many different situations [1]. However, this question also bears on the mRNA-specific forms of regulation. Historically, most examples of this phenomenon have come from oocytes and early embryonic development, situations in which transcriptional quiescence demands that variability in patterns of gene expression must be directed by posttranscriptional events. Early work provided evidence for global changes in translation of maternal mRNAs, and the past decade has seen the identification of many mRNAs that are much more specifically regulated, including those mentioned in previous sections, such as c-mos, moskar, nanos, hunchback and lin-14. Now, there are suggestions that translational control might prove to be much more common than has been widely appreciated. Direct support for this claim is largely anecdotal, obtained from the characterization of individual mRNAs and the demonstration that their translation is regulated. Among other types of supporting evidence two are notable, although for different reasons. The first addresses the extent of translational control and concerns the existence of alternative forms of translation initiation [43]. The standard textbook form of cap-dependent initiation represents only one option, and others have been identified through the characterization of viral mRNAs and their expression. One such mechanism is based on the use of internal ribosome entry sites (IRESes) — functional elements that can substitute for the 5′ cap structure in recruiting translational components. These IRESes have different requirements (sometimes dramatically different [44]) for initiation factors, and this constitutes a form of translational regulation. However, the extent to which IRESes are used by cellular mRNAs has been difficult to estimate. A solution to this problem, making use of DNA chip technology, has recently been introduced. Transcript association with polysomes, which is indicative of high translational activity (the unusual cases described above notwithstanding), was compared between two cellular RNA samples: one from control human cells and the other from cells in which cap-dependent initiation was inhibited by infection with poliovirus [45••]. A significant fraction, about 3%, of the cellular mRNAs remained in the polysomal fractions, and subsequent tests with two of these mRNAs confirmed that they include IRESes. Thus, this form of translational regulation appears to affect a large number of mRNAs. Another new example of translational regulation is notable because it implicates this form of gene regulation in the
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function of the nervous system. There is now substantial evidence implicating protein synthesis in synaptic function and, in particular in the use-dependent modifications of synaptic efficacy that underlie memory [46,47]. Recent advances argue for synapse specificity in this requirement [48•], consistent with the notion that stimulation at a particular synapse will lead to local translation in its dendritic compartment and that the proteins synthesized will have long-term effects on synaptic strength. Given this type of mechanism, it might be advantageous to have some specificity in the proteins synthesized after stimulation, suggesting that mRNA-specific forms of translational regulation may be in operation. Support for this idea has come from the demonstration that CPEB is present at expected regions in the mammalian brain and that an mRNA involved in synaptic plasticity displays CPEB-dependent translation, at least when expressed ectopically in a Xenopus oocyte [49]. For neurobiologists the most interesting questions are likely to focus on the identities of the regulated mRNAs, and the roles played by the proteins they encode. However, in terms of the impact of this work on translational control, the key point — that translation is regulated — has already been made, and the question it raises is where will the next exciting example appear?
Conclusions There is a growing understanding of the details of several different forms of mRNA-specific translational regulation. Not surprisingly, control appears to be exerted at a variety of steps in translation. Some of the best understood examples in which initiation of translation is regulated involved changes in the poly(a) tail, but this is not a universal feature. Regulatory intervention in the elongation phase of translation is suggested by recent work from flies and worms, and again the underlying mechanisms display diversity, with different types of regulators — either protein or RNA — implicated. The view that translational regulation is most widespread, and most important, in early development may be qualified in future as additional examples of this form of regulated gene expression are discovered.
Acknowledgements Work in my lab on translational regulation is supported by National Institutes of Health Grant GM54409.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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de Moor CH, Richter JD: Cytoplasmic polyadenylation elements mediate masking and unmasking of cyclin B1 mRNA. EMBO J 1999, 18:2294-2303. CPEB is shown to act not only in activation of translation, but also in repression of mRNAs bearing a CPE, the CPEB binding site.
18. Lipshitz HD, Smibert CA: Mechanisms of RNA localization and translational regulation. Curr Opin Genet Dev 2000, 10:476-488. 19. Lie Y, Macdonald PM: Translational regulation of oskar mRNA • occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts. Development 1999, 126:4989-4996. These papers [19•–21•] report the development of in vitro translation systems from Drosophila ovaries and embryos that recapitulate several examples of translational regulation. These systems should prove to be useful for defining the mechanisms of repression. Lie and Macdonald [19•] demonstrate the utility of their system and show that Bruno-dependent repression of oskar mRNA does not involve either the 5′ cap or the 3′ poly(A) tail. Gebauer et al. [20•] show that Sxl-dependent repression of msl2 mRNA occurs independently of the poly(A) tail.
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Mendez R, Hake LE, Andresson T, Littlepage LE, Ruderman JV, Richter JD: Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 2000, 404:302-307. Phosphorylation of a specific residue of CPEB is shown to be essential for polyadenylation and translation of c-mos mRNA in Xenopus oocytes and for maturation of oocytes in response to progesterone. The kinase responsible for this phosphorylation is Eg2. These results show how a signaling event controls a translational control mechanism. 10. Mendez R, Murthy KG, Ryan K, Manley JL, Richter JD: •• Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex. Mol Cell 2000, 6:1253-1259. The Eg2-dependent phosphorylation of CPEB is shown to enhance its interaction with a subunit of the cleavage and polyadenylation specificity factor (CPSF), one of several factors required for cytoplasmic polyadenylation. Using purified proteins in an in vitro system, this phosphorylation is shown to mediate the recruitment of CPSF by CPEB and the subsequent polyadenylation of bound mRNAs. These results demonstrate the molecular function of CPEB in translational activation. 11. Dickson KS, Bilger A, Ballantyne S, Wickens MP: The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation. Mol Cell Biol 1999, 19:5707-5717. 12. Kuge H, Richter JD: Cytoplasmic 3′′ poly(A) addition induces 5′′ cap ribose methylation: implications for translational control of maternal mRNA. EMBO J 1995, 14:6301-6210. 13. Tarun SZ, Sachs AB: Association of the yeast poly(A) tail binding protein with translation initation factor elF-4G. EMBO J 1996, 15:7168-7177. 14. Tarun SZ Jr, Wells SE, Deardorff JA, Sachs AB: Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proc Natl Acad Sci USA 1997, 94:9046-9051. 15. Zelus BD, Giebelhaus DH, Eib DW, Kenner KA, Moon RT: Expression of the poly(A)-binding protein during development of Xenopus laevis. Mol Cell Biol 1989, 9:2756-2760. 16. Gray NK, Coller JM, Dickson KS, Wickens M: Multiple portions of •• poly(A)-binding protein stimulate translation in vivo. EMBO J 2000, 19:4723-4733. See annotation [17••]. 17. ••
Wakiyama M, Imataka H, Sonenberg N: Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation. Curr Biol 2000, 10:1147-1150. These papers [16••,17••] provide evidence that poly(A)-binding protein (PABP) contributes to translation in Xenopus oocytes, despite expectations that the low level of the protein would preclude such a role. Wakiyama et al. showed that a mutant form of eIF4G that was unable to interact with PABP reduces translation of polyadenylated mRNAs and inhibits maturation of the oocytes. Gray et al. [16••] tethered PABP to mRNAs using a foreign RNAbinding domain and showed that bound PABP stimulated translation. Although this approach does not address the issue of PABP levels in the oocyte, it does demonstrate that the machinery necessary to respond to PABP is present.
22. Kim-Ha J, Kerr K, Macdonald PM: Translational regulation of oskar mRNA by bruno, an ovarian RNA binding protein, is essential. Cell 1995, 81:403-412. 23. Carrera P, Johnstone O, Nakamura A, Casanova J, Jackle H, Lasko P: •• VASA mediates translation through interaction with a Drosophila yIF2 homolog. Mol Cell 2000, 5:181-187. Vasa, an RNA helicase required for translation of oskar and gurken mRNAs, interacts both genetically and physically with the Drosophila homolog of IF2/eIF5B. IF2/eIF5 has been shown by others to mediate joining of the ribosomal subunits and may have other functions [24,25]. This work provides an example of how regulatory proteins can interact with the translation apparatus and should prove instrumental in defining the mechanism of Vasa action. 24. Liang L, Diehl-Jones W, Lasko P: Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities. Development 1994, 120:1201-1211. 25. Pestova TV, Lomakin IB, Lee JH, Choi SK, Dever TE, Hellen CU: The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 2000, 403:332-335. 26. Lee JH, Choi SK, Roll-Mecak A, Burley SK, Dever TE: Universal conservation in translation initiation revealed by human and archaeal homologs of bacterial translation initiation factor IF2. Proc Natl Acad Sci USA 1999, 96:4342-4347. 27.
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28. Wharton RP, Sonoda J, Lee T, Patterson M, Murata Y: The pumilio RNA-binding domain is also a translational regulator. Mol Cell 1998, 1:863-872. 29. Sonoda J, Wharton RP: Recruitment of Nanos to hunchback mRNA • by Pumilio. Genes Dev 1999, 13:2704-2712. These papers [29•,30•] reveal two options for interactions of Puf and Nanos protein family members with RNA targets. Sonoda and Wharton [28•] show that the Drosophila Nanos protein forms a ternary complex with Pumilio protein (a Puf family member) and translational regulatory sequences from the hunchback mRNA, the NREs. Formation of the complex appears to be essential for repression of hunchback mRNA, explaining the requirement for Nanos in this process. Kraemer et al. [30•] find that, in contrast, the C. elegans Nanos-3 and FBF (Puf family) can interact independently of the RNA target, in this case the fem-3 mRNA. 30. Kraemer B, Crittenden S, Gallegos M, Moulder G, Barstead R, • Kimble J, Wickens M: NANOS-3 and FBF proteins physically interact to control the sperm-oocyte switch in Caenorhabditis elegans. Curr Biol 1999, 9:1009-1018. See annotation [29•]. 31. Zhang B, Gallegos M, Puoti A, Durkin E, Fields S, Kimble J, Wickens MP: A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature 1997, 390:477-484.
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32. Smibert CA, Lie YS, Shillinglaw W, Henzel WJ, Macdonald PM: • Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro. RNA 1999, 5:1535-1547. These papers [31•,32•] report the cloning of smaug, which encodes a protein that binds to the SREs in the nanos mRNA 3′ UTR to repress its translation. Smibert et al. [31•] describe an in vitro assay that recapitulates Smaug-dependent repression, which will be useful for establishing the underlying mechanism. Dahanukar and Wharton [32•] report the isolation of a smaug mutant, which will facilitate future genetic analysis of repression and describe an interaction of Smaug with Oskar protein, which may be involved in the activation of nanos translation. 33. Dahanukar A, Walker JA, Wharton RP: Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila. Mol Cell 1999, 4:209-218. 34. Crucs S, Chatterjee S, Gavis ER: Overlapping but distinct RNA elements control repression and activation of nanos translation. Mol Cell 2000, 5:457-467. 35. Gavis ER, Lehmann R: Translational regulation of nanos by RNA localization. Nature 1994, 369:315-318. 36. Clark IE, Wyckoff D, Gavis ER: Synthesis of the posterior • determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism. Curr Biol 2000, 10:1311-1314. Evidence is presented to show that nanos mRNA is associated with polyribosomes even when no Nanos protein accumulates, suggesting that repression of nanos translation occurs at the elongation phase of protein synthesis. 37. •
Markesich DC, Gajewski KM, Nazimiec ME, Beckingham K: bicaudal encodes the Drosophila beta NAC homolog, a component of the ribosomal translational machinery. Development 2000, 127:559-572. This paper shows that the product of the bicaudal gene is the Drosophila homolog of beta NAC, a subunit of the nascent polypeptide-associated complex that binds to proteins as they emerge from the translating ribosome. bicaudal mutants allow ectopic Nanos protein accumulation, raising the possibility that the Bicaudal protein blocks nanos mRNA translation during elongation. However, the effect of Bicaudal on nanos mRNA has not yet been shown to be direct. 38. Lee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993, 75:843-854.
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42. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, •• Maller B, Hayward DC, Ball EE, Degnan B, Muller P et al.: Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000, 408:86-89. The short let-7 RNA, whose physical properties and presumed mode of action are similar to those of the lin-4 RNA, is shown to be highly conserved among animal species. In C. elegans let-7 controls the timing of certain developmental events and may perform a similar role in other animals. It also seems likely that its mechanism will be conserved, and future analyses in some of these other animals may complement the work with C. elegans. 43. Jackson RJ: A comparative view of initiation site selection mechanisms. In Translational Control of Gene Expression. Edited by Sonenberg N, Hershey JWB, Mathews MB. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2000:127-183. 44. Wilson JE, Pestova TV, Hellen CU, Sarnow P: Initiation of protein synthesis from the A site of the ribosome. Cell 2000, 102:511-520. 45. Johannes G, Carter MS, Eisen MB, Brown PO, Sarnow P: •• Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray. Proc Natl Acad Sci USA 1999, 96:13118-13123. DNA chips are used to profile polysomal mRNAs when most cap-dependent initiation of translation is inhibited. About 3% of cellular mRNAs remain associated with polysomes, revealing that cap-independent modes of translational initiation are not limited to viral or extremely rare cellular mRNAs. The different forms of translational initiation represent a type of mRNA-specific translational control. 46. Martin KC, Casadio A, Zhu H, Yaping E, Rose JC, Chen M, Bailey CH, Kandel ER: Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 1997, 91:927-938. 47.
Martin KC, Barad M, Kandel ER: Local protein synthesis and its role in synapse-specific plasticity. Curr Opin Neurobiol 2000, 10:587-592.
48. Huber KM, Kayser MS, Bear MF: Role for rapid dendritic protein • synthesis in hippocampal mGluR-dependent long-term depression. Science 2000, 288:1254-1257. Evidence is provided in this paper to show that the requirement for local translation following synaptic stimulation is synapse specific. Thus, protein synthesized in response to stimulation can affect the properties of the individual synapse, which may be the basis for long-term alterations such as those involved in memory.
39. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G: The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000, 403:901-906.
49. Wu L, Wells D, Tay J, Mendis D, Abbott MA, Barnitt A, Quinlan E, Heynen A, Fallon JR, Richter JD: CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 1998, 21:1129-1139.
40. Wightman B, Ha I, Ruvkun G: Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993, 75:855-862.
50. Lie YS, Macdonald PM: Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation. Development 1999, 126:1129-1138.
41. Olsen PH, Ambros V: The lin-4 regulatory RNA controls • developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol 1999, 216:671-680. The C. elegans lin-4 gene product is a short cytoplasmic RNA that is complementary to the 3′ UTR sequences of several regulated mRNAs, including that of lin-14. The lin-4-dependent downregulation of Lin-14 protein accumulation is shown to occur post-transcriptionally. Association of lin-14 mRNA with polysomes is similar at developmental stages before and during the phase of lin-4 action. These results indicate that the presumed interaction of lin-4 RNA with lin-14 mRNA affects the elongation phase of translation or, seemingly less likely, the stability of Lin-14 protein.
51. Gunkel N, Yano T, Markussen FH, Olsen LC, Ephrussi A: Localization-dependent translation requires a functional interaction between the 5′′ and 3′′ ends of oskar mRNA. Genes Dev 1998, 12:1652-1664. 52. Wreden C, Verrotti AC, Schisa JA, Lieberfarb ME, Strickland S: Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA. Development 1997, 124:3015-3023. 53. Bergsten SE, Gavis ER: Role for mRNA localization in translational activation but not spatial restriction of nanos RNA. Development 1999, 126:659-669.
Diversity in translational regulation Paul Macdonald Translational control of individual mRNAs relies on cis-regulatory elements, which are often found in the 3′ untranslated region. The best characterized of these regulate cytoplasmic polyadenylation, and much of this process can now be defined in terms of molecular interactions, protein modifications and their consequences. Biochemical and genetic approaches have advanced the understanding of the many instances of translational regulation that are crucial for body patterning in Drosophila. For example, in vitro translation systems have been used to study the regulatory mechanisms, and genetic interactions have been instrumental in establishing a link between a regulatory factor and a component of the translational apparatus. Although most examples of control are thought to affect the initiation of translation, two classes of regulatory factors, one a protein and one a short non-coding RNA now appear to inhibit protein synthesis during elongation. Diversity seems to be a central feature of translational control, both in the mechanisms themselves and in the situations where this form of regulation is used. Addresses Institute for Cellular and Molecular Biology, Section of Molecular Cell and Developmental Biology, The University of Texas at Austin, 2500 Speedway, Austin, Texas 78712-1095, USA; e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:326–331 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations IRESes internal ribosome entry sites PABP poly(A)-binding protein UTR untranslated region
Introduction Control of gene expression persists after transcription, when a diverse spectrum of processes that impact upon the distribution or activity of mRNAs comes into play. The most fundamental of these, given the existence of mRNAs as an intermediate in making proteins from genes, is translation. A decision of whether to translate an mRNA may be applied non-specifically, through global effects on the translation machinery. One of the most important and extensively characterized examples centers on the limiting initiation factor eIF4E, a focal point for regulation in a wide range of cell types [1]. Alternatively, features of individual mRNAs can impose mRNA-specific forms of control. In this review I describe recent progress in understanding translational regulation, focusing on the control mechanisms which are tailored to act on specific mRNAs. In most of the examples described here, the relevant elements of translational regulation are contained within the 3′ untranslated region (UTR), but despite this similarity, the ways in which these elements influence translation can differ substantially (Figure 1).
Translational control and cytoplasmic polyadenylation Many maternal mRNAs are transcribed long before they are translated. For at least some of these mRNAs, such as the c-mos mRNA of Xenopus, the onset of translation plays a crucial role in development of the oocyte [2,3]. For c-mos, and some other mRNAs, translational activation is accompanied by cytoplasmic polyadenylation [4]. This process involves the interplay between a regulatory element (the CPE) in the 3′ UTR of affected mRNAs [4], CPEB, a protein that binds to CPEs [5], and other factors. Oddly enough, CPEB mediates both repression and activation of translation [6••], and recent progress in defining the changing interactions of CPEB has provided an explanation for its opposing behaviors [7•]. During repression CPEB binds to Maskin, a protein isolated as a binding partner for CPEB. Maskin, in turn binds to the translation initiation factor eIF4E (the factor that binds specifically to the 5′ cap). Inclusion of eIF4E in this complex prevents its interaction with eIF4G, another component of the cap-binding complex. This interaction is required to bring the 40S ribosomal subunit to the mRNA. A lingering question concerns the inhibition of eIF4E: what prevents repression from acting in trans, with Maskin at the 3′ UTR of one mRNA molecule inactivating eIF4E bound to the cap of another mRNA? This issue is common to many examples of 3′ UTR-mediated translational regulation, and the answer probably lies in the nature of mRNA packaging in the cytoplasm, which may ensure that the two ends of a single mRNA are in close proximity to each other (circularization is possible under some circumstances [8]) or possibly packaged with coregulated mRNAs in particles. Reversal of CPEB-mediated repression is triggered by phosphorylation of CPEB at Ser174 by the Eg2 kinase [9••]. This change in charge enhances binding of CPEB to the component of the protein complex that adds poly(A) tails to mRNAs [10••], strengthening earlier evidence implicating this complex in cytoplasmic polyadenylation events [11]. Thus, the modification of CPEB recruits the polyadenylation machinery and elongates the poly(A) tail. This leads to the question: how does polyadenylation activate translation? Disruption of the Maskin–eIF4E complex takes place at the same time as elongation of the poly(A) tail, but how this happens is not known. Two answers have been proposed for further stimulation of translation. One invokes a 5′ cap modification that occurs in response to elongation of the poly(A) tail and enhances translation efficiency during oocyte maturation [12]. The second relies on an additional set of molecular interactions defined using in vitro experimental systems. Long poly(A) tails are bound efficiently by the poly(A)-binding protein (PABP). Because PABP binds to eIF4G, the association of PABP with mRNAs bearing long poly(A) tails can help to recruit eIF4G for binding with eIF4E at the 5′ cap and thus
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Figure 1
(a) Repression c-mos mRNA
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Current Opinion in Cell Biology
Variations in mechanisms of translational repression by factors that bind to specific control elements in 3′ UTRs of regulated mRNAs. (a) Control of c-mos translation, both repression and activation, requires CPEB. Different interactions of CPEB and Maskin determine whether translation is allowed. In this example, activation involves extension of the poly(A) tail. See text for details. (b) Repression of oskar mRNA by Bruno does not involve either the cap or the poly(A) tail, and so Bruno (Bru) must inhibit some other step, such as assembly of the complete ribosome on the mRNA [19•]. Additional 3′ UTR-binding proteins, including Apontic (Apt) [50] and possibly p50 [51], contribute to repression. (c) Repression by proteins of the Puf and Nanos families. For the Drosophila hunchback mRNA, the Puf family member Pumilio and Nanos bind to the regulatory sequences (NREs) in a ternary complex, with the Pumilio–Nanos interaction only detected in the presence of the NREs [29•]. In contrast, the C. elegans FBF (fem-3-binding factor) and Nanos-3 proteins, which repress translation of the fem-3 mRNA, can bind to one another independently of the RNA [30]. Other Nanos family members in C. elegans do not detectably interact with FBF in the absence of the
RNA but are required for the same developmental process, a switch from spermatogenesis to oogenesis in hermaphrodites. Thus, it is possible that Puf–Nanos interactions other than Pumilio–Nanos can occur only in the presence of the RNA control elements. Repression of hunchback mRNA by Pumilio and Nanos is correlated with deadenylation [52], although the details of how this effect leads to inhibition of translation for this mRNA are not known. (d) Control at the level of elongation. The Drosophila nanos mRNA is repressed by the binding of Smaug (Smg), and possibly another protein, to the 3′ UTR [32•,33,53], whereas C. elegans lin-14 mRNA is repressed through a probable interaction between its 3′ UTR and the lin-4 regulatory RNA [38–40]. In both cases repression does not substantially alter the fraction of the mRNA associated with polyribosomes [35•,40•]. In Drosophila, the Bicaudal (Bic) protein, which appears to be required for repression of nanos mRNA translation, is the homolog of a component of the nascentpolypeptide-associated complex [37•], and thus may contribute to repression through binding to nascent Nanos protein, again suggesting that repression occurs during elongation.
promote translation [13,14]. However, the low level of PABP in Xenopus oocytes [15] casts doubt on the relevance of the eIF4G–PABP interaction in this setting. Two recent reports reopen this issue. In one set of experiments PABP was targeted to specific mRNAs in Xenopus oocytes using a foreign RNA-binding domain and cognate binding site [16••]. The interaction stimulated translation, apparently through binding
to eIF4G, demonstrating the potential for a stimulatory role of PABP on endogenous mRNAs. In the second set of experiments, expression in Xenopus oocytes of a mutant form of eIF4G unable to interact with PABP had a modest dominant inhibitory effect on translation of polyadenylated mRNAs, including c-mos, and a more substantial effect on oocyte maturation [17••]. Thus, there is now evidence supporting a role
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for the poly(A) tail in recruiting PABP for stimulation of c-mos translation, and it may be that polyadenylation contributes to translational activation through multiple pathways.
Translational control as a means to restrict spatial patterns of protein expression Translational control events limit not only the timing of maternal mRNA expression but also where certain mRNAs are allowed to be translated. This phenomenon is most striking in oogenesis and early embryogenesis of Drosophila. Multiple translational regulatory events help to restrict the spatial expression of proteins that act as patterning determinants and to organize the body pattern of the embryo. The number of mRNAs and proteins known to be involved is now quite large, and a complete description of these events is beyond the scope of this review (see [18] for a recent and comprehensive account). Instead, I will highlight a few recent advances that are likely to have a significant impact on future work or resolve open issues. One recent advance is the development of Drosophilabased in vitro translation systems that reproduce translational regulation using exogenous mRNAs [19•–21•]. These systems contain all the required regulatory factors and offer the promise of dissecting translational control events at the biochemical level. Two of these reports [19•,20•] focus on the repression of oskar mRNA by Bruno, an essential regulatory event that helps ensure that the Oskar protein will be present at high levels only at the posterior pole of the oocyte [22], and the in vitro system has been used to show that repression involves neither the 5′ cap nor the poly(A) tail [19•]. A second advance is the demonstration of a functional interaction between Vasa and the translation factor IF2 [23••]. Vasa is an RNA helicase [24] required for the efficient translation of multiple proteins during oogenesis, including Oskar and Gurken [18]. IF2, also known as eIF5B, is required for joining of the ribosomal subunits [25] and may have additional functions during translation [26]. The Vasa–IF2(eIF5B) interaction provides, at last, a compelling link in flies between a genetically defined regulatory molecule and a general translation factor. Exploring and extending the Vasa–IF2(eIF5B) interaction should provide significant new insights into how regulatory molecules affect the functioning of the translation apparatus. A third notable advance concerns the translational repression of hunchback mRNA, an essential step in posterior body patterning. Nanos, the genetically defined posterior patterning determinant, has long been known to act in this example of repression [27], but its role has been obscure, with no evidence of a direct interaction with either the NREs (the sequences in hunchback mRNA that mediate repression) or Pumilio, the protein that binds specifically to the NREs [28]. Now all three components — Nanos, Pumilio and the NREs — have been shown to form a ternary complex, with the RNA component being required
to bring the two proteins together [29•]. This result contrasts with a similar situation in Caenorhabditis elegans, where two proteins closely related to Nanos and Pumilio interact with each other independently of RNA [30•]. Proteins related to Pumilio, the Puf family [31], are present in organisms ranging from yeast to humans, and it remains to be seen which, if either, of the fly and worm models will serve as the prototype for Puf interactions elsewhere. A final example from Drosophila embryogenesis involves the nanos mRNA, whose translation is repressed throughout much of the embryo by binding of Smaug protein to SREs (or Smaug recognition elements) in the nanos 3′ UTR [32•,33]. A distinct element is also involved and presumably binds to an as yet unidentified factor [34]. Translation only occurs at the posterior pole in a process dependent on a number of genes, including oskar and vasa [35]. Surprisingly, the presence of nanos mRNA in polysomal fractions persists even in oskar and vasa mutants [36•]. Thus, repression appears to be exerted after initiation of translation. Notably, a similar model had already been proposed on the basis of the characterization of the bicaudal gene [37•]. bicaudal mutants allow ectopic translation of nanos mRNA, presumably because of defects in repression of nanos translation. The bicaudal gene encodes a subunit of the nascent-polypeptide-associated complex, which binds to polypeptides as they emerge from the ribosome. Thus, if failure of the complex to bind to the nascent Nanos protein is responsible for the defect in repression, then repression must occur during elongation.
Heterochrony in C. elegans Intervention during the elongation phase of translation has also been implicated in a peculiar form of regulation highlighted by certain heterochronic mutants of C. elegans. Heterochronic mutants affect the timing of developmental events during larval stages of growth. Two of the heterchronic genes, lin-4 and the recently identified let-7, do not themselves encode proteins [38,39]; instead, their products are short cytoplasmic RNAs, each of which is largely complementary to sequences in the 3′ UTRs of overlapping sets of other heterochronic genes (such as lin-14). Where tested, loss of the complementary sequences mimics the effects of mutating the short RNAs, leading to reduced levels of the proteins encoded by the target mRNAs [38–40]. The effect on protein levels cannot be explained by altered mRNA stability, and so this appears to be a novel form of translational regulation. Recent studies have addressed the question of which translational step is the target for lin-4-dependent repression of lin-14 translation [42•]. Results very similar to those obtained with nanos mRNA, showed that repression is achieved with no obvious loss of polysomal association. Thus, the short RNAs appear to affect translation after initiation or to affect the stability of the nascent protein. Remarkably, one of the short RNAs, let-7, is present in a wide range of animal species [41••]. The timing of expression of the let-7 RNA in some of these species suggests a conserved role in temporal control of developmental events,
Diversity in translational regulation Macdonald
and it seems likely that a molecular function in translational control should also be conserved.
How widespread is translational regulation? Discussions of translational regulation often turn to the question of whether its use is limited to certain cell types or whether it has a broader role. At one level the answer is already clear, as control mechanisms that govern the translational machinery have been shown to act in many different situations [1]. However, this question also bears on the mRNA-specific forms of regulation. Historically, most examples of this phenomenon have come from oocytes and early embryonic development, situations in which transcriptional quiescence demands that variability in patterns of gene expression must be directed by posttranscriptional events. Early work provided evidence for global changes in translation of maternal mRNAs, and the past decade has seen the identification of many mRNAs that are much more specifically regulated, including those mentioned in previous sections, such as c-mos, moskar, nanos, hunchback and lin-14. Now, there are suggestions that translational control might prove to be much more common than has been widely appreciated. Direct support for this claim is largely anecdotal, obtained from the characterization of individual mRNAs and the demonstration that their translation is regulated. Among other types of supporting evidence two are notable, although for different reasons. The first addresses the extent of translational control and concerns the existence of alternative forms of translation initiation [43]. The standard textbook form of cap-dependent initiation represents only one option, and others have been identified through the characterization of viral mRNAs and their expression. One such mechanism is based on the use of internal ribosome entry sites (IRESes) — functional elements that can substitute for the 5′ cap structure in recruiting translational components. These IRESes have different requirements (sometimes dramatically different [44]) for initiation factors, and this constitutes a form of translational regulation. However, the extent to which IRESes are used by cellular mRNAs has been difficult to estimate. A solution to this problem, making use of DNA chip technology, has recently been introduced. Transcript association with polysomes, which is indicative of high translational activity (the unusual cases described above notwithstanding), was compared between two cellular RNA samples: one from control human cells and the other from cells in which cap-dependent initiation was inhibited by infection with poliovirus [45••]. A significant fraction, about 3%, of the cellular mRNAs remained in the polysomal fractions, and subsequent tests with two of these mRNAs confirmed that they include IRESes. Thus, this form of translational regulation appears to affect a large number of mRNAs. Another new example of translational regulation is notable because it implicates this form of gene regulation in the
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function of the nervous system. There is now substantial evidence implicating protein synthesis in synaptic function and, in particular in the use-dependent modifications of synaptic efficacy that underlie memory [46,47]. Recent advances argue for synapse specificity in this requirement [48•], consistent with the notion that stimulation at a particular synapse will lead to local translation in its dendritic compartment and that the proteins synthesized will have long-term effects on synaptic strength. Given this type of mechanism, it might be advantageous to have some specificity in the proteins synthesized after stimulation, suggesting that mRNA-specific forms of translational regulation may be in operation. Support for this idea has come from the demonstration that CPEB is present at expected regions in the mammalian brain and that an mRNA involved in synaptic plasticity displays CPEB-dependent translation, at least when expressed ectopically in a Xenopus oocyte [49]. For neurobiologists the most interesting questions are likely to focus on the identities of the regulated mRNAs, and the roles played by the proteins they encode. However, in terms of the impact of this work on translational control, the key point — that translation is regulated — has already been made, and the question it raises is where will the next exciting example appear?
Conclusions There is a growing understanding of the details of several different forms of mRNA-specific translational regulation. Not surprisingly, control appears to be exerted at a variety of steps in translation. Some of the best understood examples in which initiation of translation is regulated involved changes in the poly(a) tail, but this is not a universal feature. Regulatory intervention in the elongation phase of translation is suggested by recent work from flies and worms, and again the underlying mechanisms display diversity, with different types of regulators — either protein or RNA — implicated. The view that translational regulation is most widespread, and most important, in early development may be qualified in future as additional examples of this form of regulated gene expression are discovered.
Acknowledgements Work in my lab on translational regulation is supported by National Institutes of Health Grant GM54409.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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de Moor CH, Richter JD: Cytoplasmic polyadenylation elements mediate masking and unmasking of cyclin B1 mRNA. EMBO J 1999, 18:2294-2303. CPEB is shown to act not only in activation of translation, but also in repression of mRNAs bearing a CPE, the CPEB binding site.
18. Lipshitz HD, Smibert CA: Mechanisms of RNA localization and translational regulation. Curr Opin Genet Dev 2000, 10:476-488. 19. Lie Y, Macdonald PM: Translational regulation of oskar mRNA • occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts. Development 1999, 126:4989-4996. These papers [19•–21•] report the development of in vitro translation systems from Drosophila ovaries and embryos that recapitulate several examples of translational regulation. These systems should prove to be useful for defining the mechanisms of repression. Lie and Macdonald [19•] demonstrate the utility of their system and show that Bruno-dependent repression of oskar mRNA does not involve either the 5′ cap or the 3′ poly(A) tail. Gebauer et al. [20•] show that Sxl-dependent repression of msl2 mRNA occurs independently of the poly(A) tail.
Stebbins-Boaz B, Cao Q, de Moor CH, Mendez R, Richter JD: Maskin is a CPEB-associated factor that transiently interacts with elF-4E. Mol Cell 1999, 4:1017-1027. The authors show that the repressive role of CPEB involves an interaction with Maskin. The CPEB–Maskin complex binds to eIF4E and prevents its interaction with eIF4G, thus blocking initiation of translation.
20. Gebauer F, Corona DF, Preiss T, Becker PB, Hentze MW: • 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 1999, 18:6146-6154. See annotation [19•].
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21. Castagnetti S, Hentze MW, Ephrussi A, Gebauer F: Control of oskar • mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development 2000, 127:1063-1068. See annotation [19•].
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Wells SE, Hillner PE, Vale RD, Sachs AB: Circularization of mRNA by eukaryotic translation initiation factors. Mol Cell 1998, 2:135-140.
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Mendez R, Hake LE, Andresson T, Littlepage LE, Ruderman JV, Richter JD: Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 2000, 404:302-307. Phosphorylation of a specific residue of CPEB is shown to be essential for polyadenylation and translation of c-mos mRNA in Xenopus oocytes and for maturation of oocytes in response to progesterone. The kinase responsible for this phosphorylation is Eg2. These results show how a signaling event controls a translational control mechanism. 10. Mendez R, Murthy KG, Ryan K, Manley JL, Richter JD: •• Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex. Mol Cell 2000, 6:1253-1259. The Eg2-dependent phosphorylation of CPEB is shown to enhance its interaction with a subunit of the cleavage and polyadenylation specificity factor (CPSF), one of several factors required for cytoplasmic polyadenylation. Using purified proteins in an in vitro system, this phosphorylation is shown to mediate the recruitment of CPSF by CPEB and the subsequent polyadenylation of bound mRNAs. These results demonstrate the molecular function of CPEB in translational activation. 11. Dickson KS, Bilger A, Ballantyne S, Wickens MP: The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation. Mol Cell Biol 1999, 19:5707-5717. 12. Kuge H, Richter JD: Cytoplasmic 3′′ poly(A) addition induces 5′′ cap ribose methylation: implications for translational control of maternal mRNA. EMBO J 1995, 14:6301-6210. 13. Tarun SZ, Sachs AB: Association of the yeast poly(A) tail binding protein with translation initation factor elF-4G. EMBO J 1996, 15:7168-7177. 14. Tarun SZ Jr, Wells SE, Deardorff JA, Sachs AB: Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proc Natl Acad Sci USA 1997, 94:9046-9051. 15. Zelus BD, Giebelhaus DH, Eib DW, Kenner KA, Moon RT: Expression of the poly(A)-binding protein during development of Xenopus laevis. Mol Cell Biol 1989, 9:2756-2760. 16. Gray NK, Coller JM, Dickson KS, Wickens M: Multiple portions of •• poly(A)-binding protein stimulate translation in vivo. EMBO J 2000, 19:4723-4733. See annotation [17••]. 17. ••
Wakiyama M, Imataka H, Sonenberg N: Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation. Curr Biol 2000, 10:1147-1150. These papers [16••,17••] provide evidence that poly(A)-binding protein (PABP) contributes to translation in Xenopus oocytes, despite expectations that the low level of the protein would preclude such a role. Wakiyama et al. showed that a mutant form of eIF4G that was unable to interact with PABP reduces translation of polyadenylated mRNAs and inhibits maturation of the oocytes. Gray et al. [16••] tethered PABP to mRNAs using a foreign RNAbinding domain and showed that bound PABP stimulated translation. Although this approach does not address the issue of PABP levels in the oocyte, it does demonstrate that the machinery necessary to respond to PABP is present.
22. Kim-Ha J, Kerr K, Macdonald PM: Translational regulation of oskar mRNA by bruno, an ovarian RNA binding protein, is essential. Cell 1995, 81:403-412. 23. Carrera P, Johnstone O, Nakamura A, Casanova J, Jackle H, Lasko P: •• VASA mediates translation through interaction with a Drosophila yIF2 homolog. Mol Cell 2000, 5:181-187. Vasa, an RNA helicase required for translation of oskar and gurken mRNAs, interacts both genetically and physically with the Drosophila homolog of IF2/eIF5B. IF2/eIF5 has been shown by others to mediate joining of the ribosomal subunits and may have other functions [24,25]. This work provides an example of how regulatory proteins can interact with the translation apparatus and should prove instrumental in defining the mechanism of Vasa action. 24. Liang L, Diehl-Jones W, Lasko P: Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities. Development 1994, 120:1201-1211. 25. Pestova TV, Lomakin IB, Lee JH, Choi SK, Dever TE, Hellen CU: The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 2000, 403:332-335. 26. Lee JH, Choi SK, Roll-Mecak A, Burley SK, Dever TE: Universal conservation in translation initiation revealed by human and archaeal homologs of bacterial translation initiation factor IF2. Proc Natl Acad Sci USA 1999, 96:4342-4347. 27.
Rongo C, Gavis ER, Lehmann R: Localization of oskar RNA regulates oskar translation and requires Oskar protein. Development 1995, 121:2737-2746.
28. Wharton RP, Sonoda J, Lee T, Patterson M, Murata Y: The pumilio RNA-binding domain is also a translational regulator. Mol Cell 1998, 1:863-872. 29. Sonoda J, Wharton RP: Recruitment of Nanos to hunchback mRNA • by Pumilio. Genes Dev 1999, 13:2704-2712. These papers [29•,30•] reveal two options for interactions of Puf and Nanos protein family members with RNA targets. Sonoda and Wharton [28•] show that the Drosophila Nanos protein forms a ternary complex with Pumilio protein (a Puf family member) and translational regulatory sequences from the hunchback mRNA, the NREs. Formation of the complex appears to be essential for repression of hunchback mRNA, explaining the requirement for Nanos in this process. Kraemer et al. [30•] find that, in contrast, the C. elegans Nanos-3 and FBF (Puf family) can interact independently of the RNA target, in this case the fem-3 mRNA. 30. Kraemer B, Crittenden S, Gallegos M, Moulder G, Barstead R, • Kimble J, Wickens M: NANOS-3 and FBF proteins physically interact to control the sperm-oocyte switch in Caenorhabditis elegans. Curr Biol 1999, 9:1009-1018. See annotation [29•]. 31. Zhang B, Gallegos M, Puoti A, Durkin E, Fields S, Kimble J, Wickens MP: A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature 1997, 390:477-484.
Diversity in translational regulation Macdonald
32. Smibert CA, Lie YS, Shillinglaw W, Henzel WJ, Macdonald PM: • Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro. RNA 1999, 5:1535-1547. These papers [31•,32•] report the cloning of smaug, which encodes a protein that binds to the SREs in the nanos mRNA 3′ UTR to repress its translation. Smibert et al. [31•] describe an in vitro assay that recapitulates Smaug-dependent repression, which will be useful for establishing the underlying mechanism. Dahanukar and Wharton [32•] report the isolation of a smaug mutant, which will facilitate future genetic analysis of repression and describe an interaction of Smaug with Oskar protein, which may be involved in the activation of nanos translation. 33. Dahanukar A, Walker JA, Wharton RP: Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila. Mol Cell 1999, 4:209-218. 34. Crucs S, Chatterjee S, Gavis ER: Overlapping but distinct RNA elements control repression and activation of nanos translation. Mol Cell 2000, 5:457-467. 35. Gavis ER, Lehmann R: Translational regulation of nanos by RNA localization. Nature 1994, 369:315-318. 36. Clark IE, Wyckoff D, Gavis ER: Synthesis of the posterior • determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism. Curr Biol 2000, 10:1311-1314. Evidence is presented to show that nanos mRNA is associated with polyribosomes even when no Nanos protein accumulates, suggesting that repression of nanos translation occurs at the elongation phase of protein synthesis. 37. •
Markesich DC, Gajewski KM, Nazimiec ME, Beckingham K: bicaudal encodes the Drosophila beta NAC homolog, a component of the ribosomal translational machinery. Development 2000, 127:559-572. This paper shows that the product of the bicaudal gene is the Drosophila homolog of beta NAC, a subunit of the nascent polypeptide-associated complex that binds to proteins as they emerge from the translating ribosome. bicaudal mutants allow ectopic Nanos protein accumulation, raising the possibility that the Bicaudal protein blocks nanos mRNA translation during elongation. However, the effect of Bicaudal on nanos mRNA has not yet been shown to be direct. 38. Lee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993, 75:843-854.
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42. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, •• Maller B, Hayward DC, Ball EE, Degnan B, Muller P et al.: Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000, 408:86-89. The short let-7 RNA, whose physical properties and presumed mode of action are similar to those of the lin-4 RNA, is shown to be highly conserved among animal species. In C. elegans let-7 controls the timing of certain developmental events and may perform a similar role in other animals. It also seems likely that its mechanism will be conserved, and future analyses in some of these other animals may complement the work with C. elegans. 43. Jackson RJ: A comparative view of initiation site selection mechanisms. In Translational Control of Gene Expression. Edited by Sonenberg N, Hershey JWB, Mathews MB. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2000:127-183. 44. Wilson JE, Pestova TV, Hellen CU, Sarnow P: Initiation of protein synthesis from the A site of the ribosome. Cell 2000, 102:511-520. 45. Johannes G, Carter MS, Eisen MB, Brown PO, Sarnow P: •• Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray. Proc Natl Acad Sci USA 1999, 96:13118-13123. DNA chips are used to profile polysomal mRNAs when most cap-dependent initiation of translation is inhibited. About 3% of cellular mRNAs remain associated with polysomes, revealing that cap-independent modes of translational initiation are not limited to viral or extremely rare cellular mRNAs. The different forms of translational initiation represent a type of mRNA-specific translational control. 46. Martin KC, Casadio A, Zhu H, Yaping E, Rose JC, Chen M, Bailey CH, Kandel ER: Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 1997, 91:927-938. 47.
Martin KC, Barad M, Kandel ER: Local protein synthesis and its role in synapse-specific plasticity. Curr Opin Neurobiol 2000, 10:587-592.
48. Huber KM, Kayser MS, Bear MF: Role for rapid dendritic protein • synthesis in hippocampal mGluR-dependent long-term depression. Science 2000, 288:1254-1257. Evidence is provided in this paper to show that the requirement for local translation following synaptic stimulation is synapse specific. Thus, protein synthesized in response to stimulation can affect the properties of the individual synapse, which may be the basis for long-term alterations such as those involved in memory.
39. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G: The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000, 403:901-906.
49. Wu L, Wells D, Tay J, Mendis D, Abbott MA, Barnitt A, Quinlan E, Heynen A, Fallon JR, Richter JD: CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 1998, 21:1129-1139.
40. Wightman B, Ha I, Ruvkun G: Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993, 75:855-862.
50. Lie YS, Macdonald PM: Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation. Development 1999, 126:1129-1138.
41. Olsen PH, Ambros V: The lin-4 regulatory RNA controls • developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol 1999, 216:671-680. The C. elegans lin-4 gene product is a short cytoplasmic RNA that is complementary to the 3′ UTR sequences of several regulated mRNAs, including that of lin-14. The lin-4-dependent downregulation of Lin-14 protein accumulation is shown to occur post-transcriptionally. Association of lin-14 mRNA with polysomes is similar at developmental stages before and during the phase of lin-4 action. These results indicate that the presumed interaction of lin-4 RNA with lin-14 mRNA affects the elongation phase of translation or, seemingly less likely, the stability of Lin-14 protein.
51. Gunkel N, Yano T, Markussen FH, Olsen LC, Ephrussi A: Localization-dependent translation requires a functional interaction between the 5′′ and 3′′ ends of oskar mRNA. Genes Dev 1998, 12:1652-1664. 52. Wreden C, Verrotti AC, Schisa JA, Lieberfarb ME, Strickland S: Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA. Development 1997, 124:3015-3023. 53. Bergsten SE, Gavis ER: Role for mRNA localization in translational activation but not spatial restriction of nanos RNA. Development 1999, 126:659-669.